EP1866651B1 - Method for analyzing a blood sample - Google Patents
Method for analyzing a blood sample Download PDFInfo
- Publication number
- EP1866651B1 EP1866651B1 EP06726120A EP06726120A EP1866651B1 EP 1866651 B1 EP1866651 B1 EP 1866651B1 EP 06726120 A EP06726120 A EP 06726120A EP 06726120 A EP06726120 A EP 06726120A EP 1866651 B1 EP1866651 B1 EP 1866651B1
- Authority
- EP
- European Patent Office
- Prior art keywords
- analysis
- sample
- tank
- dilution
- solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y10T436/25—Chemistry: analytical and immunological testing including sample preparation
- Y10T436/25625—Dilution
Definitions
- the present invention relates to a method for the analysis of a blood sample.
- the invention more particularly relates to the field of automatic analyzes of blood samples.
- Each circuit is characterized by a dilution rate of the blood sample adapted to the measurement means used, the addition of one or more reagents and appropriate means of implementation and measurement.
- the circuit typically comprises a so-called counting tank in which the blood sample is diluted, a reagent comprising in particular the red blood cell lysing compound is added thereto, the stabilization compound of the hemoglobin complex and the leucoprotective compound, and hemoglobin is measured directly in this vessel by spectrophotometry and the number of white blood cells by resistivity.
- the dilution ratio is chosen so that the analysis solution is perfectly homogeneous and so that the detection apparatus is not saturated. This dilution rate is between 1 / 100th and 1/500 generally between 1/160 th and 1/180 th.
- the circuit uses a dilution vat of the blood sample in which one or more reagents containing a lysing agent of the red blood cells, optionally a differentiation agent (for example a fluorescent dye of the DNA or RNA of the white blood cells), and then a fraction of this solution to inject it into an optical flow cell of a flow cytometer.
- a differentiation agent for example a fluorescent dye of the DNA or RNA of the white blood cells
- the dilution rate is less than 1 / 100th , making it possible to obtain, with the currently known cytometers on the market (hydrofocus type), an optimum analysis time.
- the main objectives of the manufacturers are to simplify existing PLCs by reducing the number of components and reagents, which reduces the manufacturing costs, maintenance and size of the automata, without reducing the time of an analysis complete with a blood sample.
- the present invention aims in particular to achieve these objectives.
- the document WO 2004/003517 proposes in this sense a method and an apparatus where the two analysis circuits have common means.
- the principle is to perform a first dilution of the blood sample in a single dilution tank and successively transfer fractions of selected volumes of this dilution to a measuring or counting unit, to measure or count each time elements different contents in the blood sample.
- the document describes the following solution: use a first transfer to count the red blood cells and the platelets, add a lysing agent in the dilution tank, then perform a second transfer to count the white blood cells, perform a third transfer of lysed dilution solution to measure the hemoglobin, add a differentiation reagent leukocyte, and perform a fourth transfer to achieve leukocyte differentiation in the measurement set.
- This principle makes it possible to implement a single so-called dilution tank, but it does not allow a gain in analysis time since the measurements or counting are performed successively after each transfer of a fraction of the dilution. In addition, it requires perfect control of the volumes of successive reagents and diluents distributed as well as dilution volumes transferred to the measurement package. It also further requires the implementation of several syringes and lysis reagents.
- US 5,958,781 A discloses a method of analyzing a blood sample using a lysis mono-reagent operating in tandem with a specific diluent for measuring hemoglobin and enumerating at least three leukocyte subpopulations, possibly by optical, although no text data supports the use of optical sensing means and the associated results. This process is based on the use of a specific diluent whose composition is indicated.
- the document US 5,786,224A discloses a method of analyzing a blood sample using a bi-reagent composition as a lysing agent, allowing the differentiation of at least three sub-leukocyte populations. This process does not involve prior dilution with a diluent.
- the document US 4529705A discloses a method of analyzing a blood sample using a single reagent for both diluting and lysing red blood cells, and containing a hemoglobin complexing agent, specifically cyanide ion (or its complex ferricyanide) whose toxicity is well established.
- a hemoglobin complexing agent specifically cyanide ion (or its complex ferricyanide) whose toxicity is well established.
- the globular enumeration proposed by this method makes it possible to quantify the total number of white blood cells but not the differentiation of the subpopulations, and only by resistive electronic means (not optical).
- the document US RE38131 E also describes a method of analyzing a blood sample using a mono-reagent for the determination of two or three leukocyte subpopulations, but only by resistive route.
- the present invention further aims to overcome such drawbacks.
- the method according to the invention is characterized in that the analysis solution also contains at least one compound for protecting the white blood cells, allowing the discrimination of the five main leukocyte subpopulations.
- the count of the white blood cells can be carried out jointly in the analysis tank and / or by the optical means.
- the count of red blood cells and possibly platelets can be carried out for example in a preliminary step of the method on a sample taken in the single dilution and analysis tray.
- the present invention is based on the concept of a single analytical solution used as such for the two types of analyzes which were traditionally conducted in two separate circuits, namely on the one hand the measurement of hemoglobin and possibly counting the white blood cells and, secondly, the leukocyte differentiation by optical means, said analysis solution bringing together the "reactive" compounds capable of carrying out at least these analyzes, by their nature and their quantity.
- the reactive compounds introduced are chosen to be chemically compatible with each other and in appropriate quantities for the targeted analyzes. They can be chosen from the compounds typically used in the prior art.
- Optical measurement means making it possible to obtain an analysis of the white blood cells (counting and differentiation by subpopulations) at a dilution ratio greater than 1/100 e are also proposed according to the invention and are defined and described below.
- the monoreactant used in the method according to the invention allows the spectrophotometric measurement of the hemoglobin concentration of a blood sample and a leukocyte differentiation by an optical means. It also allows resistive and / or optical counting of white blood cells. Preferably, it is chosen to allow the differentiation of at least 5 subpopulations. Preferably, it is chosen so as not to contain cyanides.
- the compound for lysing red blood cells is preferably composed of at least one cationic surfactant.
- Component Quantity Cationic surfactant 0,1-50g / L Leucoprotective surfactant 0,1-20g / L Chelate of the hemoglobin complex 0,0001-10g / L Dye 0,01-1g / L Fixative agent 0.01-2% w / v Wetting agent 0-50% v / v Buffer 0-6g / L Bottom salt 1-50g / L Acid Amount suitable for setting the pH Tory Appropriate amount 0.1-3g / L ionophore Effective amount 0-200mg / L Distilled water qs qsp 1L
- the present disclosure is directed to an optical device for an automaton for automatic analysis of a blood sample, which is particularly advantageous for the implementation of the method according to the first subject of the invention.
- certain leukocyte subpopulations can be differentiated only by optical measurements, for example a diffraction measurement by the cell at one or more angles, or a measurement of the absorbance of the cell.
- Optical characterization systems of a blood cell have a common base in which we find a light source emitting a beam light, an optical tank in which the blood cells pass through the light beam, a light beam adjustment system on the flow of cells and means for measuring the light from the optical tank after interception by the cells.
- the white blood cells move in a flow in the tank. They are illuminated by a light beam focused on the flow, called sample flow.
- Such devices are expensive; in particular, lasers used as light sources, which are also bulky and generally require a heat dissipation system; Laser diodes, like lasers, require expensive alignment systems.
- the light beams emitted by these sources have a transverse distribution of the light of substantially Gaussian form.
- the intensity is substantially constant and maximum only in a narrow and central part of the radius. Alignment systems align this central portion with the sample flow.
- the width of the sample flow must not exceed that of this central part, and the accuracy of the alignment system must be even greater than these two widths are close. This results in the need to minimize the width of the sample stream.
- the sample flow containing the blood cells to be counted and / or to be differentiated must be narrower the closer the light is focused.
- a flux whose section width is less than 50 .mu.m which must pass through the light beam itself focused in a brush section greater than that of the sample stream.
- This requires a particularly precise injection system, therefore expensive, the flow in the optical tank.
- hydrofocus-type system contraction of the English expression "hydrodynamic focusing"
- the sample flow is sheathed by a sleeve flow.
- An injector for the sample flow is immersed in the core of the sleeve flow.
- the sample stream thus created is stretched, or focused, during its journey from the injector to the area illuminated by the light beam, so that it has a desired width of about 5 to 50 ⁇ m in diameter. A single or double sleeve is sometimes necessary to achieve this goal.
- an adjustment system is essential to make the flow of cells coincide with the light beam.
- Two approaches are possible: we can move the cell flow or the light beam. If you choose to move the flow of blood cells, you must move the entire optical tank. When this option is chosen, the tank is mounted on a translation table which ensures a smooth and uniform movement along two axes thanks to its ball bearings. Such a mechanical set of precision is quite expensive. It is also possible to move the light beam to coincide with the flow of blood cells. This is usually done using several orientable prisms. This solution, which combines optical elements with precision mechanics, also involves a high cost.
- the blood cell deviates the trajectory of the light rays.
- the intensity and the angle of the deviated rays make it possible to obtain information on the type of the cell.
- Two ranges of angles are generally used: small angles less than ten degrees from the optical axis and large angles substantially perpendicular to the optical axis. In the range of small angles, two pieces of information are useful: the losses in the axis and the diffraction. Perpendicular to the optical axis, diffusion and fluorescence are generally measured.
- the light must be distributed in two different channels. This is usually done by dichroic mirrors or by interference filters. These optical components are both made by deposition of thin layers on a glass substrate. Their efficiency is good but there is a great disparity from one filter to another and their life is limited. They must therefore be changed regularly.
- the purpose of the disclosure is to propose a leucocyte differentiation device and / or leucocyte counting that is simpler and more economical both to manufacture and to maintain, allowing the use of automated systems as well as equipped, by laboratories of less importance, while keeping a sufficient quality of measurement.
- an optical device for leukocyte counting and / or differentiation in a blood analysis machine characterized in that it comprises a light-emitting diode-type light source for illuminating a blood sample which circulates in the tank. optical along an injection axis, using a source light beam.
- a light-emitting diode-type light source for illuminating a blood sample which circulates in the tank. optical along an injection axis, using a source light beam.
- the diode emits light whose wavelength is less than 600 nanometers, and even more preferably less than 500 nanometers.
- a wavelength allows a better diffraction efficiency, thus a better accuracy for measurements using diffraction.
- the width of the beam emitted by the optical device is advantageously between 50 and 200 microns ( ⁇ m), in the vicinity of the injection axis. This allows a larger sample flow to be illuminated, while allowing sufficient precision to the measurements made. Even more advantageously, this width is between 90 and 120 microns. Such a flux width is notably allowed by the use of light emitting diodes.
- the source light beam is emitted substantially in the direction of the tank, substantially transversely to the flow direction of the sample.
- a transparent blade designed to be traversed between two opposite faces by the source beam, mounted in rotation and disposed between the diode and the tank can allow to move transversely the light beam, thanks to its double refraction during the crossing of the blade.
- the rotation of the blade makes it possible to modify the angle of incidence of the beam on the blade, and thus to adjust the value of the transverse offset.
- the transparent blade is rotated about an axis substantially parallel to the displacement of the blood sample in the tank.
- Fresnel loss separation means are advantageously used for a resulting light beam. incident from the source light beam, thereby separating said beam into an axial resultant beam, and at least one resultant beam of loss constituted by Fresnel losses at the passage of the separation means.
- the separation means comprise at least one separation surface which is a surface of a transparent separation material, the axial beam having passed through the transparent material and the beam resulting from Fresnel losses having been reflected by the separation surface, said surface being biased relative to the light beam beyond the tank.
- a simple glass slide, inexpensive can serve as a means of separation. In addition, it has an almost unlimited and maintenance-free life, unlike dichroic mirrors or interference filters.
- the device may further comprise an apparatus for measuring the light of the resulting axial beam and at least one other apparatus for measuring the light of the at least one beam resulting from Fresnel losses.
- These measurement devices can in particular comprise means for a measurement among a measurement of the fluorescence, a measurement of the light losses in the vicinity of the axis and a measurement of the diffraction in the vicinity of the axis. It may further comprise means for measuring the diffraction of the light beam at large angles by the sample in the tank. For example, these large angles may be angles between 60 ° and 150 °.
- the device may also comprise, in the path of the beam before the tank, at least one stop diaphragm for stray light.
- the disclosure also relates to a hematology apparatus, in particular a blood analysis machine equipped with such a device.
- the present disclosure also relates to a circulating optical tank for an optical device suitable for leucocyte counting and differentiation, for example a flow cytometer, as well as an analysis apparatus equipped with such a tank.
- the object of the invention is to provide a simpler and more economical tank both to manufacture and maintain, allowing the use of automated machines and equipped by smaller laboratories, while maintaining a sufficient measurement quality.
- a flow cell for an optical device for leukocyte counting and differentiation in an automated analyzer characterized in that in an analysis zone of the vessel (304), the section of the vessel has at least one transverse dimension of between 1 and 5 millimeters.
- This section may be substantially rectangular and the transverse dimension be measured on one and / or the other side of the rectangle.
- Such a tank can then be made, at least partially, of an injected plastic material.
- Such a tank is particularly advantageous to manufacture relative to the tanks of the prior art, generally formed of quartz walls assembled by gluing.
- the tank may further comprise at least one lens molded integrally with the tank.
- This at least one lens may comprise a lens designed to be disposed laterally relative to an optical axis. It can include a hemispherical lens.
- the tank may comprise, along an optical axis, a window for the entry of a light beam and a window for the exit of the beam.
- At least one window may be molded in one piece with a tank and / or be an insert made of a transparent material, for example quartz or glass.
- the vessel may advantageously comprise an injector for a sample flow and means for forming a sleeving flow around the injection flow.
- the injector may comprise an outlet orifice whose diameter is between 20 microns and 150 microns, making it possible to obtain a sample flow that is substantially larger than the fluxes of the prior art. Unlike the devices of the prior art, it is not the sleeving flow that imposes the width of the sample flow by stretching it, but the shape and the outlet section of the injector. The sheath flow therefore no longer has an active role, but only a passive role, in particular, for example, to center the sample flow in a tank of great width.
- this injector can be formed in one piece from a substantially rigid material.
- This material may be, for example, a stainless steel, a ceramic, synthetic ruby or a plastic or many of these materials.
- this injector may comprise a tube of rigid structure, for example a metal structure, for example made of stainless steel, and inside the structural tube, a plastic cladding tube extended by a spout formed of one piece with the sheath tube.
- the plastic material of the injector may be a polytetrafluoroethylene, which allows easier movement of the sample in the tube and reduces the risk of fouling.
- the disclosure also relates to an injector for a tank according to the disclosure, an injector made according to one of these embodiments.
- the disclosure also relates to a hematology apparatus, in particular a blood analysis machine, equipped with a tank according to the invention.
- the present disclosure also relates to a hydraulic device for a hematological analysis device, which is simplified and more economical both to manufacture and to maintain and which allows the use of automated devices equipped with such a device by laboratories of lesser importance. , while keeping a sufficient quality of measurement.
- the present disclosure also relates to an analysis method adapted to such a device.
- the present disclosure thus proposes a hydraulic device for a blood analysis apparatus, in particular an automaton, comprising means for injecting under pressure a stream of sample into a circulating optical tank and to create a liquid sleeve flow around the flow of the sample.
- sample with a sleeving liquid, characterized in that it comprises means for regulating a flow rate of the sample flow relative to the flow rate of the sleeving liquid.
- Such a regulation can make it possible to maintain homogeneous and substantially non-turbulent flows in the tank.
- the injection means may comprise syringes, a hydraulic circuit and solenoid valves. These means may comprise means for injecting the sample under pressure relative to the sleeve flow.
- This device may advantageously comprise means for forming a piston for the sample injected with a liquid piston.
- a sweeping liquid makes it possible to use only a small sample sufficient for the analysis, the remainder of the liquid necessary for the injection being a liquid available in the analysis apparatus, and less valuable than the sample.
- the device may advantageously comprise means for regulating a flow rate of the sweeping liquid relative to the flow rate of the sleeving liquid.
- the regulating means may comprise means of pressure drop in a bypass circuit for the piston liquid and / or pressure loss means in a bypass circuit for the sleeving liquid.
- the pressure drop means may be chosen from a known length of a calibrated pipe, a fixed hydraulic resistance and a variable resistance.
- the hydraulic device may comprise only one motor, for example a single electric motor, to simultaneously generate the sample flow and the sleeve flow.
- it may comprise at least two syringes to generate the sample flow and the sleeve flow, the syringe pistons being rigidly interconnected. They are thus animated by a common movement and the sample and sleeving flows are simultaneous.
- a hydrofocus tank of the prior art can be used with a circuit as previously described, the injection of the sample into this tank can be done without pressure relative to the sleeving fluid.
- a method of analyzing a blood sample in a flow cytometer characterized in that a blood sample is injected, possibly under pressure, into a flow cell of the cytometer, the sample forming a sample flow and creating a liquid sleeve flow around the sample stream, with a sleeving liquid, characterized in that the flow rate of the sample flow is regulated relative to the flow of the sleeving liquid .
- This slurry liquid may be selected from a reagent and a diluent, preferably a reagent. It is therefore not necessary to provide another than those strictly necessary for the preparation of the sample for its analysis.
- a sleeve flow with a sleeving liquid can also be selected from a reagent and a diluent, preferably a diluent. Again, it is not necessary to provide a liquid other than those strictly necessary for the preparation of the sample for his or her analysis.
- the blood sample has a dilution ratio of at least 1 / 100th. Indeed, in such a method, it is possible to introduce the sample under pressure relative to the sleeving fluid, into the tank, at a speed greater than that of the processes of the prior art, and with widths of higher sections for the flow. sample in the tank. Thus, without increasing the analysis time, it is possible for differentiation and counting of white blood cells to use a dilution ratio identical to that customarily used for the measurement of hemoglobin, in particular dilution levels substantially between 1 / 100 th and 1/500 th, particularly between 1/160 th and 1/180 th.
- the disclosure also relates to a hematology apparatus, particularly a blood analysis machine, characterized in that it comprises a hydraulic device according to the invention.
- FIG. 1 is schematized a single vat of dilution and analysis that can be fed by a blood sample 2 to be analyzed, a diluent 3 and a reagent 4 together forming an analysis solution.
- This bin 1 is equipped with photometry measuring means 5 of the hemoglobin level in said analysis solution and means 6 for measuring the resistivity of said analysis solution for counting the global number of white blood cells.
- Means are also provided for taking a fraction of the analysis solution in the analysis tank 1 and for injecting it into an optical tank 7 equipped with optical measuring means 8 (for example a flow cytometer) for an analysis of the white blood cells.
- means are further provided for taking a fraction of a pre-solution consisting of the sample of blood and diluent, and introducing it into a counting and dilution tank 9 provided with measuring the resistivity of said fraction to count red blood cells and platelets.
- the equipment is provided with heating means to obtain a thermostatically controlled temperature of substantially 35 ° C. This temperature allows for optimal lysis reaction time and lysis quality of red blood cells.
- An optical device particularly suitable for leucocyte analysis on an analysis solution having a dilution ratio of less than 1/100 e is described later, more particularly adapted to a dilution between 1/160 e and 1/180 e .
- a dilution ratio of 1/160 e is lower than a rate of 1/100 e .
- the tray 1 can be used in a second time to count red blood cells and platelets after cleaning, filling the tray with a sample pending in a needle of a syringe.
- FIGS. 2f to 2i are cytographs obtained using a BD FACScan® flow cytometer, corresponding respectively to Eosinofix alone, Eosinofix to which DDAPS, Tiron and imidazole are added.
- FIG. 2f it can be seen that the differentiation of subpopulations of white blood cells is well carried out and comparable to a conventional leucocyte differentiation reagent (matrix obtained with Eosinofix at figure 2f ).
- the figure 3 is a partial diagram of a hydraulic circuit 100 and some equipment of a blood analysis machine, provided that they allow the understanding of the hydraulic device.
- the automaton illustrated at figure 3 includes a needle 101 for collecting blood to be analyzed in a tube used for storage and transport to the controller.
- the blood taken is poured in the form of a sample by the needle in a tray 102.
- the tray 102 is especially intended for the dilution and / or the lysis of the red blood cells of the blood sample. All or part of the sample, before or after dilution, can be taken for analysis in another part of the automaton, for example in a device 120, described later.
- a device for analyzing hemoglobin 110 (for example a spectrophotometer) is arranged in the vicinity of the tank 102.
- a reserve 103 for a dilution product and a reserve 104 for a reagent, in particular for lysis, are connected to the tank 102 via the hydraulic circuit 100.
- Another analysis device 120 is more particularly dedicated to the counting and differentiation of white blood cells, for example on all or part of the sample taken from the tray 102. Thereafter we will also call this sample all or this part.
- the device for analyzing the white blood cells 120 comprises in particular an optical device 200 and an optical tank 300.
- the optical tank is connected to the tank 102 via the hydraulic circuit 100.
- a syringe set allows the movement of liquids in the hydraulic circuit.
- a syringe 105 dedicated to the diluent and a syringe 106 dedicated to the reagent are shown for a good understanding of the device.
- the hydraulic circuit comprises solenoid valves for switching different circuits in the hydraulic circuit 100, according to the use made of it at a given moment of the analysis.
- Eight solenoid valves 111-119 among solenoid valves of the hydraulic circuit 100 are illustrated in FIG. figure 3 .
- Each solenoid valve comprises two positions, each marked respectively by the letter A or B.
- the design of the hydraulic circuit as will be described later allows to use only one motorization M for syringes illustrated.
- the same motorization can be used for other syringes.
- the pistons of the syringes 105, 106 are rigidly interconnected. They therefore have a simultaneous movement, either in thrust P, when they sink into the respective cylinder of each syringe, or in traction T when they are extracted.
- the vessel 300 comprises an outer body 301 and an injector 302, within the body 301, a sleeve volume 303 is formed between the body and the injector.
- the diluting syringe 105 is in communication with the diluent reservoir, so that a pull T can fill the syringe 105 with diluent.
- a thrust P can move the diluent to this use 108, for example in the tray 102, for example for a dilution of the overall sample.
- a thrust P makes it possible to move the diluent into the optical tank 300, to form a sleeving flow.
- the utility of this sleeving flow in the context of the invention will be analyzed in a subsequent description of the tank 300.
- valve 117 Since the valve 117 is in a first position 117A which connects the reagent syringe with the reagent reservoir 104, and the valve 114 is in a first position 114A which cuts the discharge branch 134, a pull T makes it possible to fill the reagent syringe. 106 with reagent.
- a thrust P makes it possible to move the reagent to this use 109, for example in the tray 102, for example for lysis of the overall sample.
- valve 117 being in its second position 117B and the valve 111 being in its second position 111B which connects the reaction branch 141 with the injection arm 131, the reagent syringe 106 is directly connected to the injector 302.
- valve 118 being in a first position 118A which isolates the suction branch 133 from the sample branch 132 through the drain branch
- valve 112 being in a first position 112A which connects the upstream to the downstream of the sample branch 132
- valve 113 being in a first position 113A which connects the downstream to the upstream of the suction branch 133, therefore to the vacuum source 107, the sample to be analyzed is sucked into the injection branch 131, between the sample connection 142 and the suction connection 143.
- the discharge branch 134 comprises a variable or calibrated fluid resistance 150.
- the reagent syringe 106 contains reagent and a blood sample to be analyzed is in the injection arm 131; when in addition the valves 112, 113 are in their second positions 112B, 113B which isolate the upstream and downstream of their respective branches; when the valves 115, 116 are in their second positions 115B, 116B which connect the diluent syringe 105 with the sleeve volume 303; when finally the valves 111, 117 are in their second positions 111B, 117B which connect the reagent syringe 106 with the injector 302 and the valve 114 is in its second position 114B; a single thrust movement P generated by the single motorization M, makes it possible to propel the diluent, the reagent and the blood sample towards and through the tank 300, while part of the reagent, depending on the fluidic resistance 150, is returned to the reagent pool
- the resistor 150 makes it possible in particular to adjust the relative flow rates of the sleeving and swab liquids. This makes it possible to adapt these flow rates to the different functions of these liquids. In particular, this makes it possible to have similar flow speeds for the sleeving and the sample in the analysis zone 304 when using a conventional hydrofocus tank.
- discharge branch 134 and the previously described arrangements allow the use of a single motor, thus significantly reducing the cost of an analysis automat, as well as its bulk.
- the diluent forms in a test zone 304 of the tank 300 a sleeve flow for the sample (see particularly the Figures 4 and 5 ).
- the reagent located upstream of the sample in the injection arm 131, serves as a swabbing liquid, that is to say, it makes it possible to transmit the movement of the plunger of the syringe of reagent to the sample . Thus, it is not useful to fill the reagent syringe with the sample for analysis. Thus, even a sample of small volume can be analyzed, and all of this sample can be injected and analyzed without a part remaining in the injection arm 131 or in the syringe 106.
- optical device 200 will now be described, particularly with regard to Figures 4 and 5 .
- the optical device comprises a source 201 of substantially monochrome light.
- This light source is a light emitting diode.
- the light is mainly emitted along an X200 optical axis.
- the optical axis X200 is disposed substantially perpendicular to an injection axis X300 of displacement of the sample in the optical tank 300.
- the two axes X200 and X300 together define an optical plane.
- a set of three diaphragms is each arranged perpendicularly on the path of the beam.
- the diaphragms 202 are pierced with orifices whose diameter is substantially equal to the beam and progressively increased in each diaphragm to adapt to the diameter of the beam as the diameter increases. away from the source 201.
- the beam then passes through a focusing device 203 consisting of one or more lenses.
- the beam encounters an adjustment device which makes it possible to move the optical axis in a plane perpendicular to the injection axis X300, that is to say transversely relative to the displacement of the sample. in the tank.
- a lateral shift of the beam can lead to partial or no illumination of the sample which has a direct influence on the result of the analysis.
- the adjustment device consists of a transparent blade 220 rotatably mounted about an axis X220.
- the axis 221 is substantially parallel to the injection axis X300. If the blade is disposed perpendicular to the optical axis X200, the beam passes through it without being deflected. On the other hand, if the blade forms an angle with the optical axis, a double reduction, at the input and at the output of the blade, shifts the beam in a plane perpendicular to the adjustment axis X220.
- the X220 adjustment axis being substantially parallel to the X300 injection axis, only a transverse offset is generated by the refraction in the blade.
- the offset is greater the greater the thickness and / or the refractive index of the blade and the blade is inclined relative to the optical axis.
- it is sufficient to rotate the blade 220 about its axis X220 to adjust the position of the beam relative to the sample that moves in the analysis zone 304 of the optical tank 300.
- Such an adjustment device is particularly economical with respect to the devices of the prior art, especially since precise rotation is generally easier to implement than accurate translation, using high accuracy.
- the source beam 211 After penetrating the vessel and passing through the sample, the source beam 211 at least partially becomes an axial resultant beam 212, which leaves the vessel substantially along the optical axis.
- the resulting axial beam 212 carries information on the sample that has just passed through.
- optical analysis relies on the detection of diffracted light according to two ranges of angles: small and large angles. In each of the ranges of angles, two different pieces of information are used. It is therefore appropriate to distribute the light in two different channels for each range.
- Means 205 are therefore used to separate the resulting beam 212 into two resulting beams 213,214.
- the separation means consist mainly of a separating blade 205. This separating blade is a transparent glass slide. It is arranged at 45 degrees of the optical axis. This results in a secondary axial resultant beam 213, formed by light having passed through the splitter plate, and a resulting loss beam 214 formed by Fresnel losses, i.e. by the light reflected by the splitter plate.
- Such a separating blade is of a very low cost relative to the separation means used in the prior art in optical analysis devices of this type. In particular, because it does not include any reflective coating added, it is almost unalterable over time and requires substantially no maintenance. Given the multiple reflections inside the plate and taking into account the polarization of the incident radiation of the resulting axial beam, between 5 and 15% of the energy is reflected, the remainder being transmitted in the form of the resulting secondary axial beam .
- the resulting axial beam 212 is made parallel by suitable means 206.
- the resulting beams 213,214 are again focused by respective adapted means 207,208, for their analysis. by the respective measuring apparatus 222,223.
- the measuring apparatus 222 which analyzes the secondary axial resultant beam 213 is a device for measuring the diffraction near the optical axis by the blood cells (so-called measurement of the FSC).
- the measuring apparatus 223, which analyzes the beam resulting from the losses of Fresnel 214 is a device for measuring the light losses in the axis (so-called measurement of the ALL), that is to say say the occultation of light by the cells in the sample.
- the figure 5 schematically represents a section of the tank in a plane perpendicular to the X300 injection axis and containing the optical axis X200. As particularly illustrated in this figure, the light reemitted laterally by the sample is also analyzed in a lateral resultant stream 315, focused beyond the tank in a measuring apparatus 224.
- the tank 350 of the figure 10 comprises a body 351, an injector 302 and an analysis zone 354.
- a transverse internal dimension D354 of the vessel is about 250 microns. This dimension may be a diameter, if the vessel is of circular section, or a side, if it is square or rectangular section.
- a sleeve flow 362 is used to significantly reduce the diameter of a sample flow 361, so that in the analysis zone 354 the sample flow has, in the prior art, a diameter D361 less than 50 microns.
- the tank 300 illustrated in Figures 4-6 , comprises the body 301 and the injector 302, arranged substantially coaxially along an injection axis X300.
- the analysis zone 304 is disposed downstream of the injector.
- the body is made of an injected material, preferably a plastic material. Such a manufacturing method makes it possible to obtain complex shapes.
- a lens 305 is molded into the body. This lens makes it possible to collect the light occulted, diffracted or diffused by the blood cells.
- This lens must have dimensions, including a sufficient diameter so that any local inhomogeneities in the injected material are negligible relative to these dimensions.
- the lens 305 has a diameter of 3 mm.
- This injected lens is a lateral lens 305 traversed by the resultant lateral beam 315.
- the lateral lens must make it possible to collect the light in the maximum of directions, that is to say with a maximum directional field. Thus, more the lens is near the sample, the wider the directional field.
- the lens is a hemispherical lens, called at 90 °.
- the lens being a part of the wall of the tank, there is direct contact with the liquid in the tank, that is to say that there is no air gap, low refractive index, between the sample and the lens. The measure is all the better.
- the light is particularly focused glass
- a glass type BK7 This is particularly the case for the axial windows 306, where the source beam 211 enters the tank and where the resulting axial beam 212 comes out.
- the tank 300 has at least comparable dimensions.
- these large dimensions make it possible to integrate glass windows in plastic walls, whereas in the tanks of the prior art, of small dimensions, these are made entirely of glass or quartz walls.
- the inner section of the tank is 4.5mm along the optical axis by 3mm in the perpendicular direction.
- This large rectangular section associated with a small volume of the sample, which carries the blood cells to be analyzed, requires the use of hydrodynamic sleeving of the sample.
- a vessel of the prior art has a transverse internal dimension D354 of the analysis zone of about 250 microns.
- the body 301 of the tank envelops the injector 302 and forms around the injector the sleeve volume 303.
- the walls of the injector separate a stream 311 formed by the sample, inside the injector with a sleeve flow 312, in the sleeve volume.
- the sample flow comes from the injection branch 131 of the hydraulic circuit 100.
- the sleeve flow is derived from the sleeve branch 135 of the hydraulic circuit.
- the two streams are in contact, remain concentric and flow simultaneously in the tank.
- a sample flow of large section will be turbulent, which would significantly affect the accuracy of measurements.
- the sample flow would be progressively reduced in section, which goes against the desired effect, which is to have a large section sample flow.
- Such a goal is achieved thanks to the implementation of the hydraulic circuit 100, previously described with reference to the figure 1 .
- Such a circuit makes it possible to obtain independently chosen speeds for the flow of the sleeving flow and for that of the sample flow, so that little turbulence appears in the sample flow and that these turbulences do not have noticeable effect on the result of the analysis.
- the two streams may each be substantially uniform, possibly laminar in certain appropriate speed ranges.
- an injector 302 as illustrated in FIG. figure 7 or at figure 8 also helps to limit turbulence in the sample flow. In addition, it allows the sample to be injected at high speed into the optical tank, while maintaining a substantially uniform flow.
- An injector 302 as illustrated in FIG. figure 7 comprises a structural tube 320, for example made of stainless steel ensuring the rigidity of the injector.
- the structural tube is sheathed internally by a tube 321 made of a plastic material, for example a polytetrafluoroethylene (PTFE).
- PTFE polytetrafluoroethylene
- the structural and sheath tubes are cylindrical.
- the cladding tube extends, downstream of the structural tube, by a spout made of the same plastic material.
- the spout has a gradually narrowed section from an inside diameter D321 of the cladding tube to an inside diameter D323 of an outlet port 323 at a downstream end 324 of the spout 322.
- the downstream end 324 is a cylinder of length L324.
- the wall of the bill is first concave inward, then bent to become internally convex, the beak section is thus gradually narrowed from upstream to downstream, from the diameter D321 to the diameter D324.
- the concave surface is tangent with the inner surface of the cylindrical cladding tube.
- the convex surface is tangent with the inner surface of the cylindrical end 324.
- the diameter D323 of the orifice 323 is about 60 microns
- the diameter D321 inside of the cladding tube is about 1 millimeter
- the length L322 of the nozzle is about 2.5 millimeters
- that L320 of the structural tube about 6 millimeters
- that of the cylindrical end L324 about 200 microns.
- An injector 302 as illustrated in Figures 8 and 9 is in one piece and a single substantially rigid material.
- This material may be, for example, stainless steel, a ceramic, a synthetic ruby or a plastic material.
- the plastics material may advantageously be polytetrafluoroethylene.
- the injector comprises a substantially cylindrical tube 331 extended downstream by a spout 332.
- the beak gradually narrows internally, from an inside diameter D331 for the tube 331, to an inside diameter D333 of an outlet 333 for the sample, at a downstream end 334 of the beak 332.
- the shrinkage is made according to a truncated cone open at an included angle, preferably between 9 and 10 degrees. Beyond the conical frustum east to the outlet orifice 333, the diameter remains constant in a cylindrical portion 335, of length L335 and of diameter D333.
- the outer diameter is progressively less reduced along an open coast trunk at an angle of between 8 and 9 degrees, then, in the much smaller end along an open coast trunk.
- D334 is about 3 to 4 larger than D333.
- D333 60 ⁇ m
- D334 200 ⁇ m
- A334 40%.
- the various devices described above it is possible to obtain a high injection speed.
- This in the example described it is possible to injected a sample of more than 200 microliters in less than 10 seconds.
- such an injection speed makes it possible to use a high dilution rate of the blood sample, without increasing the duration of the analysis relative to prior art automata.
- the same dilution for example 1 / 160th, can be used for the analysis of hemoglobin by the device 110 (see figure 3 ) and for the analysis of white blood cells by the optical device 120, instead of 1 / 80th generally used for the analysis of white blood cells.
- the figures 11a-c illustrate the results obtained by implementing the method according to the invention, and the apparatus said apparatus using an optical tank 7 and an optical device 8.
- the figure 11a shows a positive linearity test of the hemoglobin measurement and thus highlights the possible and reliable measurement of the hemoglobin level of a blood sample according to the invention.
- the figure 11b shows an optical matrix obtained on a 30% eosinophil blood test sample in which the formulation was added according to the method of the invention. On this matrix, the five sub-populations are indeed present and differentiated (sets delimited on the cytograph: E for eosinophils, N for neutrophils, M for monocytes, B for basophils and L for lymphocytes).
- the figure 11c shows the positive linearity test on resistivity measurement of white blood cell count.
- a fluidic resistor can be disposed on the sleeve circuit or both. This can be done as a function of the maximum flow rate given by the liquid displacement means intended respectively for swaging or sleeving.
- the lenses of the optical tank and / or the optical device may also be made injected with the body of the tank, instead of one as previously illustrated.
- the glass windows can be injected.
- the inhomogeneities in the injected material are substantially negligible compared to the accuracy required for measurements.
- An adjusting device and / or the separating means described above may be used independently of one another and possibly with another light source than a light-emitting diode.
Description
La présente invention concerne un procédé pour l'analyse d'un échantillon de sang. L'invention vise plus particulièrement le domaine des analyses automatiques d'échantillons de sang.The present invention relates to a method for the analysis of a blood sample. The invention more particularly relates to the field of automatic analyzes of blood samples.
On recherche par l'analyse d'un échantillon de sang à déterminer généralement :
- le nombre total de globules blancs ;
- plus spécifiquement, le nombre de globules blancs par sous populations (basophiles, éosinophiles, neutrophiles, monocytes et lymphocytes) ;
- le nombre de globules rouges et de plaquettes ; et
- le taux d'hémoglobine.
- the total number of white blood cells;
- more specifically, the number of white blood cells per subpopulation (basophils, eosinophils, neutrophils, monocytes and lymphocytes);
- the number of red blood cells and platelets; and
- the hemoglobin level.
Plusieurs techniques d'analyse sont connues, notamment :
- le dosage de l'hémoglobine est effectué après la lyse des globules rouges, c'est-à-dire la destruction de la membrane des cellules de globules rouges, et par mesure par spectrophotométrie de l'hémoglobine libérée dans le milieu ; le dosage de l'hémoglobine nécessite en outre la stabilisation de l'hémoglobine sous une forme complexée (oxyhémoglobine ou cyanméthémoglobine) de manière à mesurer l'absorbance d'un seul composé à la longueur d'onde appropriée.
- le comptage total des globules blancs est effectué sur l'échantillon de sang par résistivité en réalisant la lyse spécifique des globules rouges et la protection des globules blancs.
- la différenciation des globules blancs et leur comptage par sous population sont effectués :
- soit par mesure volumétrique en résistivité après lyse spécifique des globules rouges, protection des globules blancs et réglage du pH ; cela ne permettant cependant pas de différentier toutes les sous populations en une seule analyse ;
- soit par voie optique, notamment par cytométrie de flux ; après lyse spécifique des globules rouges et protection des globules blancs, en mesurant différents paramètres (notamment diffraction, fluorescence, absorbance) sur un flux de globules blancs dans l'axe au petits, moyens et grands angles et éventuellement après ajout d'un agent de marquage (par exemple du noir de chlorazol, ou un colorant marquant l'ADN ou l'ARN, ou un colorant fluorescent) et par mesure à différentes longueurs d'onde ; cette technique permettant de différentier les sous-populations de globules blancs.
- le comptage des globules rouges et des plaquettes se fait sur un échantillon dilué sans ajout de réactif particulier par mesure de résistivité.
- the determination of hemoglobin is carried out after the lysis of the red blood cells, that is to say the destruction of the membrane of the cells of red blood cells, and by spectrophotometric measurement of the hemoglobin released into the medium; the determination of hemoglobin also requires the stabilization of hemoglobin in a complexed form (oxyhemoglobin or cyanmethemoglobin) so as to measure the absorbance of a single compound at the appropriate wavelength.
- the total count of white blood cells is performed on the blood sample by resistivity by performing the specific lysis of red blood cells and the protection of white blood cells.
- the differentiation of white blood cells and their counting by subpopulation are carried out:
- either by volumetric measurement in resistivity after specific lysis of the red blood cells, protection of the white blood cells and adjustment of the pH; however, this does not differentiate all subpopulations in one analysis;
- either optically, in particular by flow cytometry; after specific lysis of red blood cells and protection of white blood cells, by measuring different parameters (in particular diffraction, fluorescence, absorbance) on a flow of white blood cells in the axis at small, medium and large angles and possibly after addition of a labeling agent (for example chlorazol black, or a dye marking the DNA or RNA, or a fluorescent dye) and by measurement at different wavelengths; this technique makes it possible to differentiate subpopulations of white blood cells.
- the count of red blood cells and platelets is done on a diluted sample without adding any particular reagent by measuring resistivity.
Il existe de nombreux automates d'hématologie qui mettent en oeuvre ces techniques afin d'obtenir une analyse la plus complète possible d'un échantillon de sang.There are many automated hematology machines that use these techniques to obtain the most complete analysis possible of a blood sample.
Dans ces automates, coexistent traditionnellement deux circuits d'analyse différents :
- un premier circuit conçu pour la mesure de l'hémoglobine et/ou le comptage total des globules blancs ; et
- un deuxième circuit conçu pour réaliser sur l'échantillon de sang la différenciation et/ou un comptage leucocytaire par cytométrie de flux.
- a first circuit designed for measuring hemoglobin and / or total count of white blood cells; and
- a second circuit designed to perform on the blood sample differentiation and / or leukocyte counting by flow cytometry.
Chaque circuit est caractérisé par un taux de dilution de l'échantillon de sang adapté aux moyens de mesure utilisés, l'ajout d'un ou plusieurs réactifs et des moyens de mise en oeuvre et de mesure appropriés.Each circuit is characterized by a dilution rate of the blood sample adapted to the measurement means used, the addition of one or more reagents and appropriate means of implementation and measurement.
Ainsi, pour la mesure de l'hémoglobine et le comptage des globules blancs, le circuit comporte typiquement une cuve dite de comptage dans laquelle on dilue l'échantillon de sang, on y ajoute un réactif comprenant notamment le composé de lyse des globules rouges, le composé de stabilisation du complexe formé de l'hémoglobine et le composé leucoprotecteur, et on mesure directement dans cette cuve l'hémoglobine par spectrophotométrie et le nombre de globules blancs par résistivité. Le taux de dilution est choisi de sorte que la solution d'analyse soit parfaitement homogène et de sorte que l'appareil de détection ne soit pas saturé. Ce taux de dilution est compris entre 1/100e et 1/500e, en général entre 1/160e et 1/180e.Thus, for measuring hemoglobin and counting white blood cells, the circuit typically comprises a so-called counting tank in which the blood sample is diluted, a reagent comprising in particular the red blood cell lysing compound is added thereto, the stabilization compound of the hemoglobin complex and the leucoprotective compound, and hemoglobin is measured directly in this vessel by spectrophotometry and the number of white blood cells by resistivity. The dilution ratio is chosen so that the analysis solution is perfectly homogeneous and so that the detection apparatus is not saturated. This dilution rate is between 1 / 100th and 1/500 generally between 1/160 th and 1/180 th.
Pour la différenciation leucocytaire par cytométrie de flux, le circuit met en oeuvre une cuve de dilution de l'échantillon de sang dans laquelle on ajoute un ou plusieurs réactif contenant un agent de lyse des globules rouges, éventuellement un agent de différenciation (par exemple un colorant fluorescent de l'ADN ou l'ARN des globules blancs), puis on prélève une fraction de cette solution pour l'injecter dans une cuve optique de circulation d'un cytomètre de flux. Ici, le taux de dilution pratiqué est inférieur à 1/100e, permettant d'obtenir avec les cytomètres connus sur le marché actuellement (de type hydrofocus) un temps d'analyse optimum.For leukocyte differentiation by flow cytometry, the circuit uses a dilution vat of the blood sample in which one or more reagents containing a lysing agent of the red blood cells, optionally a differentiation agent (for example a fluorescent dye of the DNA or RNA of the white blood cells), and then a fraction of this solution to inject it into an optical flow cell of a flow cytometer. Here, the dilution rate is less than 1 / 100th , making it possible to obtain, with the currently known cytometers on the market (hydrofocus type), an optimum analysis time.
Ainsi, traditionnellement, au moins deux réactifs différents doivent être utilisés pour les deux circuits d'analyse et deux dilutions différentes de l'échantillon de sang sont pratiquées dans ces deux circuits d'analyse.Thus, traditionally, at least two different reagents must be used for the two analysis circuits and two different dilutions of the blood sample are made in these two analysis circuits.
Les objectifs principalement visés par les fabricants sont de simplifier les automates existants en réduisant le nombre de composants et de réactifs, cela permettant de réduire les coûts de fabrication, de maintenance et la taille des automates, sans pour autant réduire les temps d'une analyse complète d'un échantillon de sang.The main objectives of the manufacturers are to simplify existing PLCs by reducing the number of components and reagents, which reduces the manufacturing costs, maintenance and size of the automata, without reducing the time of an analysis complete with a blood sample.
La présente invention tend notamment à atteindre ces objectifs.The present invention aims in particular to achieve these objectives.
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Ce principe permet certes de mettre en oeuvre une seule cuve dite de dilution, mais il ne permet pas un gain du temps d'analyse puisque les mesures ou comptage sont réalisés successivement après chaque transfert d'une fraction de la dilution. En outre, il nécessite un contrôle parfait des volumes de réactifs successifs et de diluants distribués ainsi que des volumes de dilution transférés vers l'ensemble de mesure. Il nécessite en outre encore la mise en oeuvre de plusieurs seringues et réactifs de lyse.This principle makes it possible to implement a single so-called dilution tank, but it does not allow a gain in analysis time since the measurements or counting are performed successively after each transfer of a fraction of the dilution. In addition, it requires perfect control of the volumes of successive reagents and diluents distributed as well as dilution volumes transferred to the measurement package. It also further requires the implementation of several syringes and lysis reagents.
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La présente invention a en outre pour objectif de pallier de tels inconvénients.The present invention further aims to overcome such drawbacks.
La présente invention vise un procédé d'analyse automatique d'un échantillon de sang, dans lequel :
- * on forme, dans un bac unique dit de dilution et d'analyse, une solution d'analyse contenant :
- ledit échantillon de sang,
- un diluant,
- un mono réactif comprenant au moins un composé pour lyser les globules rouges, au moins un composé pour stabiliser l'hémoglobine sous forme d'oxyhémoglobine et au moins un composé pour protéger les globules blancs, permettant la discrimination d'au moins cinq principales sous-populations leucocytaires, ledit composé pour protéger les globules blancs étant choisi parmi les bétaïnes et sulfo-bétaïnes d'ammoniums quaternaires, les oxydes d'aminés tertiaires, les composés de type glycosidiques et les composés de type glucidiques,
- * on mesure sur cette solution d'analyse dans ledit bac après la lyse des globules rouges le taux d'hémoglobine par spectrophotométrie ; et
- * on prélève une quantité appropriée de cette solution d'analyse dans ledit bac sur laquelle on réalise une différenciation leucocytaire d'au moins 5 sous-populations par un moyen optique.
- * in a single tray called dilution and analysis, an analysis solution containing:
- said blood sample,
- a diluent,
- a mono-reagent comprising at least one compound for lysing red blood cells, at least one compound for stabilizing hemoglobin in the form of oxyhemoglobin and at least one compound for protecting white blood cells, allowing the discrimination of at least five major subunits; leukocyte populations, said compound for protecting white blood cells being selected from quaternary ammonium betaines and sulfobetaines, tertiary amine oxides, glycosidic compounds and carbohydrate-type compounds,
- on this analysis solution in said tray after the lysis of red blood cells the hemoglobin level is measured by spectrophotometry; and
- an appropriate quantity of this analysis solution is taken from said tray on which a leukocyte differentiation of at least 5 subpopulations is carried out by optical means.
Le procédé selon l'invention est caractérisé en ce que la solution d'analyse contient en outre au moins un composé pour protéger les globules blancs, permettant la discrimination des cinq principales sous-populations leucocytaires.The method according to the invention is characterized in that the analysis solution also contains at least one compound for protecting the white blood cells, allowing the discrimination of the five main leukocyte subpopulations.
Le comptage des globules blancs peut être réalisé conjointement dans le bac d'analyse et/ou par le moyen optique.The count of the white blood cells can be carried out jointly in the analysis tank and / or by the optical means.
Le comptage des globules rouges et éventuellement des plaquettes peut être réalisé par exemple dans une étape préalable du procédé sur un prélèvement effectué dans le bac unique de dilution et d'analyse.The count of red blood cells and possibly platelets can be carried out for example in a preliminary step of the method on a sample taken in the single dilution and analysis tray.
Ainsi, la présente invention est basée sur le concept d'une solution unique d'analyse utilisée telle quelle pour les deux types d'analyses qui étaient traditionnellement menées dans deux circuits séparés, à savoir d'une part la mesure de l'hémoglobine et éventuellement le comptage des globules blancs et, d'autre part, la différenciation leucocytaire par un moyen optique, ladite solution d'analyse réunissant les composés « réactifs » capables de mener à bien au moins ces analyses, de par leur nature et leur quantité. Les composés réactifs introduits sont choisis pour être chimiquement compatibles entre eux et en quantités adaptées aux analyses visées. Ils peuvent être choisis parmi les composés typiquement utilisés dans l'art antérieur. On peut aussi utiliser une formulation commerciale utilisée classiquement pour réaliser une différentiation leucocytaire, c'est-à-dire contenant le composé pour lyser les globules rouges et le composé leucoprotecteur, et lui ajouter le troisième composé réactif destiné à stabiliser l'hémoglobine sous la forme d'un complexe chromogène.Thus, the present invention is based on the concept of a single analytical solution used as such for the two types of analyzes which were traditionally conducted in two separate circuits, namely on the one hand the measurement of hemoglobin and possibly counting the white blood cells and, secondly, the leukocyte differentiation by optical means, said analysis solution bringing together the "reactive" compounds capable of carrying out at least these analyzes, by their nature and their quantity. The reactive compounds introduced are chosen to be chemically compatible with each other and in appropriate quantities for the targeted analyzes. They can be chosen from the compounds typically used in the prior art. It is also possible to use a commercial formulation conventionally used to carry out a leucocyte differentiation, that is to say containing the compound for lysing the red blood cells and the leucoprotective compound, and to add to it the third reagent compound intended to stabilize the hemoglobin under the form of a chromogenic complex.
Grâce à cette solution d'analyse unique, la présente invention présente notamment les avantages suivants :
- l'automate peut comporter un bac unique de préparation de la solution d'analyse,
- la mesure de l'hémoglobine peut être réalisée directement sur ce bac, ainsi que le comptage global des globules blancs par mesure de résistivité de la solution d'analyse ;
- on peut mettre en oeuvre un monoréactif réunissant tous les composés « réactifs » nécessaires pour la mesure de l'hémoglobine et pour la différenciation leucocytaire par un moyen optique ; ceci permet notamment de simplifier les circuits hydrauliques comme on le verra plus loin ;
- on peut réaliser une mono-dilution de l'échantillon de sang directement dans le bac unique de dilution et d'analyse, avec un taux de dilution déterminé en fonction des moyens de mesure et de détection utilisés. Le mono-réactif peut servir de diluant pour réaliser cette monodilution. De préférence, on fixera un taux de dilution compris
entre 1/100e et 1/500e, correspondant au taux de dilution nécessaire à une mesure du taux d'hémoglobine, de préférence encore un tauxenviron de 1/175e (1/173e dans l'exemple de réalisation donné plus loin).
- the automaton can comprise a single tray for preparing the analysis solution,
- measurement of hemoglobin can be performed directly on this tray, as well as the global count of white blood cells by resistivity measurement of the analysis solution;
- a mono-reagent combining all the "reagent" compounds necessary for the measurement of hemoglobin and for leucocyte differentiation by an optical means can be used; this makes it possible in particular to simplify the hydraulic circuits as will be seen later;
- it is possible to perform a mono-dilution of the blood sample directly in the single dilution and analysis tank, with a dilution rate determined according to the measurement and detection means used. The mono-reagent can serve as a diluent to achieve this monodilution. Preferably, set a dilution ratio of between 1 / 100th and 1 / 500th, corresponds to the rate of dilution necessary to measurement of hemoglobin, more preferably a rate of about 1/175 e (1/173 e in the exemplary embodiment given below).
Avec la possibilité de mettre en oeuvre une mono-dilution et un mono-réactif, on peut donc, grâce à ce premier aspect de l'invention, simplifier grandement les appareillages d'analyse tout en offrant toujours la possibilité de réaliser une analyse complète de l'échantillon de sang.With the possibility of using a mono-dilution and a mono-reagent, it is therefore possible, thanks to this first aspect of the invention, to greatly simplify the analysis apparatus while still offering the possibility of carrying out a complete analysis of the blood sample.
Des moyens de mesure optique permettant d'obtenir une analyse des globules blancs (comptage et différenciation par sous populations) à un taux de dilution supérieur à 1/100e sont proposés également selon l'invention et sont définis et décrits plus loin.Optical measurement means making it possible to obtain an analysis of the white blood cells (counting and differentiation by subpopulations) at a dilution ratio greater than 1/100 e are also proposed according to the invention and are defined and described below.
Le monoréactif utilisé dans le procédé selon l'invention permet la mesure par spectrophotométrie de la concentration en hémoglobine d'un échantillon de sang et une différentiation leucocytaire par un moyen optique. Il permet en outre le comptage résistif et/ou optique des globules blancs. De manière préférentielle, il est choisi pour permettre la différenciation d'au moins 5 sous-populations. De manière préférentielle, il est choisi de façon à ne pas contenir de cyanures.The monoreactant used in the method according to the invention allows the spectrophotometric measurement of the hemoglobin concentration of a blood sample and a leukocyte differentiation by an optical means. It also allows resistive and / or optical counting of white blood cells. Preferably, it is chosen to allow the differentiation of at least 5 subpopulations. Preferably, it is chosen so as not to contain cyanides.
Selon l'invention, le composé pour lyser les globules rouges est de préférence constitué d'au moins un surfactant cationique.According to the invention, the compound for lysing red blood cells is preferably composed of at least one cationic surfactant.
Dans la présente invention on choisit de former un complexe oxyhémoglobine (car non toxique par comparaison à un complexe cyanméthémoglobine mettant en oeuvre des ions cyanures). Le surfactant cationique est donc choisi de manière en outre à oxyder l'hémoglobine libérée pour ne former qu'un complexe oxyhémoglobine. La quantité de surfactant cationique est donc choisie de manière à hémolyser efficacement les globules rouges et oxyder l'hémoglobine libérée. Il est de préférence choisi parmi :
- les sels d'ammoniums quaternaires, de préférence les sels d'alkyltriméthylammoniums et plus particulièrement encore les bromures et chlorures de cétyl-, dodécyl-, tétradécyl- et hexadécyltriméthylammonium ;
- les sels de pyridiniums ;
- les amines à longues chaînes éthoxylées ; et
- les alkylsulfates (SDS).
- quaternary ammonium salts, preferably alkyltrimethylammonium salts and more particularly cetyl-, dodecyl-, tetradecyl- and hexadecyltrimethylammonium bromides and chlorides;
- pyridinium salts;
- ethoxylated long chain amines; and
- alkyl sulphates (SDS).
Le composé leucoprotecteur selon l'invention est un composé retardant ou prévenant la destruction des globules blancs. De préférence, il s'agit d'un surfactant non ionique ou amphotère choisi de préférence parmi :
- les alcools éthoxylés, notamment le 2-phénoxyéthanol, les polyoxyéthylènealkylphényléther, comme les produits commercialisés IPEGAL990®, TERGITOL NP9®, TRITON® X100 ou X114, plurafac® A38 ou Brij35®) ;
- les bétaïnes et sulfo-bétaïnes d'ammoniums quaternaires notamment la lauramidopropyl bétaïne (LAB), et le dodécyldiméthyl-3-ammonio-1-propanesulfonate (DDAPS) ou le tétradécyldiméthyl-3-ammonio-1-propanesulfonate (TDAPS) ;
- les oxydes d'amines tertiaires, comme le N,N-diméthyllaurylamine-N-oxyde (LDAO) ou le sulfonate de 3-[(3cholamidopropyl)-diméthylamino-]-1-propane (CHAPS ou CHAPSO) ;
- les composés de type glycosidiques et plus particulièrement une saponine triterpénique ;
- les composés de type glucidiques (mannitol, D-glucose, tréhalose, sulfate de dextran).
- ethoxylated alcohols, in particular 2-phenoxyethanol, polyoxyethylenealkylphenylether, as the products sold IPEGAL990 ®, Tergitol NP9 ®, Triton ® X100 or X114, Plurafac A38 ® or Brij35 ®);
- quaternary ammonium betaines and sulfobetaines, in particular lauramidopropyl betaine (LAB), and dodecyldimethyl-3-ammonio-1-propanesulphonate (DDAPS) or tetradecyl dimethyl-3-ammonio-1-propanesulphonate (TDAPS);
- tertiary amine oxides, such as N, N-dimethyllaurylamine-N-oxide (LDAO) or 3 - [(3cholamidopropyl) -dimethylamino] -1-propane sulfonate (CHAPS or CHAPSO);
- compounds of the glycosidic type and more particularly a triterpene saponin;
- carbohydrate-type compounds (mannitol, D-glucose, trehalose, dextran sulfate).
Le composé stabilisant l'hémoglobine sous la forme d'un complexe chromogène d'oxyhémoglobine est de préférence choisi parmi :
- les chélates mono ou polydendates présentant des atomes ligands (doublets non liants : O, N, S, et groupements carboxy COO-...) notamment :
- les sels de l'acide éthylènediaminetétraacétique (EDTA) ou de l'acide éthylèneglycol-bis-(3-aminoéthyléther)N-N'-tétraacétique (EGTA) et notamment leurs sels disodiques ou dipotassiques ;
- l'oxalate de potassium K2Ox Ox=C2O4 2- ; et
- les acides organiques.
- les composés aromatiques (chélates mono ou polydendates) comportant des atomes ligands (présentant des doublets non liants : O, N, S...), notamment :
- le Tiron® ;
- la pyridine ou bipyridine et leurs dérivés ;
- les composés phénoliques (mono ou bis et leurs dérivés) ;
- le pyrazole et/ou les pyrazolones et leurs dérivés ;
- l'imidazole et ses dérivés ;
- l'acide sulfosalicylique ; et
- les saponines, les oxydes d'amines tertiaires, les bétaïnes et sulfo-bétaïnes d'ammoniums quaternaires (comme le DDAPS, TDAPS, LAB).
- the mono or polydendate chelates having ligand atoms (non-linking doublets: O, N, S, and carboxy groups COO -...), in particular:
- salts of ethylenediaminetetraacetic acid (EDTA) or of ethyleneglycol-bis- (3-aminoethylether) N-N'-tetraacetic acid (EGTA) and in particular their disodium or dipotassic salts;
- potassium oxalate K 2 O x O x = C 2 O 4 2- ; and
- organic acids.
- aromatic compounds (mono or polydendate chelates) comprising ligand atoms (having non-linking doublets: O, N, S, etc.), in particular:
- Tiron®;
- pyridine or bipyridine and their derivatives;
- phenolic compounds (mono or bis and their derivatives);
- pyrazole and / or pyrazolones and their derivatives;
- imidazole and its derivatives;
- sulfosalicylic acid; and
- saponins, tertiary amine oxides, quaternary ammonium betaines and sulfo-betaines (such as DDAPS, TDAPS, LAB).
En outre des trois composés définis selon l'invention, on peut ajouter au(x) (mono)réactif(s) :
- au moins un colorant (ou mélange) marquant spécifiquement certains leucocytes et plus particulièrement les éosinophiles (ou basophiles), pour permettre de discriminer au moins les 5 principales sous-populations leucocytaires, choisi parmi :
- les cyanines ;
- l'Oxazine 750 ;
- les réactifs de Wright et Romanowsky ;
- le DAPI ;
- le Noir Chlorazole E ;
- le bleu de Toluidine ;
- l'Astra Blue ;
- et le thiazole orange G. ou blue ;
- d'autres réactifs fluorescents.
- au moins un agent fixateur permettant de rigidifier la membrane des globules blancs qui sera préférentiellement un aldéhyde et plus particulièrement le glutaraldéhyde ou formaldéhyde ;
- au moins un agent mouillant pour optimiser la fluidique et éviter la formation de bulles agissant en outre comme agent de solubilisation des débris, choisi parmi :
- les alcools (méthanol éthanol ou propan-2-ol) ;
- les glycols (éthylène ou propylène glycol) ;
- les alcools éthoxylés (particulièrement le Triton X100® ou le Brij35®) ;
- les composés glycosidiques TWEEN80® ou TWEEN20® ;
- un système tampon pour fixer le
5,0 et 10,0 et de préférence entre 6,0pH entre et 8,0 et de façon optimale proche de la neutralité (7,0±0,4). Le choix d'un tel pH vise à respecter les conditions natives des cellules. En outre ce pH permet une meilleure dissolution des composants mis en oeuvre selon l'invention. Ledit tampon est constitué d'un couple de sels (inorganiques ou organiques) amené au pH précité par de l'acide chlorhydrique ou de la soude (4-6N), choisi parmi :- le dihydrogénophosphate/hydrogénophosphate H2PO4 -/HPO4 2- de sodium ou potassium ;
- l'hydrogénocarbonate/carbonate de sodium NaHCO3 /Na2CO3
- un tampon acide citrique/citrate de sodium (III)
- le TRIS-HCl
- la triéthanolamine (TEA)
- l'imidazole
- un acide choisi parmi :
- les acides organiques : phtalique, sulfosalicylique ou formique qui contribuent également à la formation et stabilisation du complexe chromogène de l'hémoglobine) ; et
- les acides minéraux : HCl, H3PO4...
- un sel de fond assurant une conductivité de l'ordre de 10 à 50ms/cm nécessaire pour la mesure de résistivité et une osmolarité de l'ordre de 120 à 500mOsm et préférentiellement proche de l'isotonicité (290±5mOsm), choisi parmi :
- le chlorure de sodium NaCl ;
- le chlorure de potassium KCl ;
- le chlorure de magnésium MgCl2 ;
- le chlorure de calcium CaCl2 ;
- le sulfate de sodium anhydre Na2SO4 ; ce sel de fond pouvant être compris dans le système tampon ;
- au moins un conservateur, ayant des propriétés antioxydantes, et/ou antibiotiques choisi parmi :
- le 2-phénoxyéthanol ;
- les parabens ;
- le BHT ;
- les isothiazolones (
Proclin® 150 ou 300) ; - les dérivés de l'imidazole ou de l'urée ;
- les antibiotiques ;
- un composé de pénétration cellulaire (ionophore) antibiotique naturel qui facilite en outre la pénétration du ou des colorants choisi parmi :
- l'ionophore I à NH4 + (Nonatine) ;
- l'ionophore III à Ca2+ (Calcimycine) ;
- l'ionophore à Cl- ;
- L'ionophore I à K+ (Valinomycine).
- at least one dye (or mixture) specifically marking certain leucocytes and more particularly eosinophils (or basophils), to discriminate at least the 5 major leukocyte subpopulations, chosen from:
- cyanines;
- Oxazine 750;
- the reagents of Wright and Romanowsky;
- the DAPI;
- the Black Chlorazole E;
- Toluidine blue;
- the Astra Blue;
- and orange thiazole G. or blue;
- other fluorescent reagents.
- at least one fixing agent for stiffening the membrane of white blood cells which will preferably be an aldehyde and more particularly glutaraldehyde or formaldehyde;
- at least one wetting agent for optimizing the fluidics and preventing the formation of bubbles further acting as a debris solubilizing agent, selected from:
- alcohols (methanol ethanol or propan-2-ol);
- glycols (ethylene or propylene glycol);
- ethoxylated alcohols (particularly Triton X100® or Brij35®);
- glycosidic compounds TWEEN80® or TWEEN20®;
- a buffer system for setting the pH between 5.0 and 10.0 and preferably between 6.0 and 8.0 and optimally close to neutral (7.0 ± 0.4). The choice of such a pH is intended to respect the native conditions of the cells. In addition, this pH allows a better dissolution of the components used according to the invention. Said buffer consists of a pair of salts (inorganic or organic) brought to the abovementioned pH by hydrochloric acid or sodium hydroxide (4-6N), chosen from:
- dihydrogen phosphate / hydrogen phosphate H 2 PO 4 - / HPO 4 2 - sodium or potassium;
- sodium hydrogencarbonate / carbonate NaHCO 3 / Na 2 CO 3
- a citric acid / sodium citrate (III) buffer
- TRIS-HCl
- triethanolamine (TEA)
- imidazole
- an acid chosen from:
- organic acids: phthalic, sulfosalicylic or formic which also contribute to the formation and stabilization of the chromogenic complex of hemoglobin); and
- the mineral acids: HCl, H 3 PO 4 ...
- a bottom salt providing a conductivity of the order of 10 to 50 ms / cm necessary for the resistivity measurement and an osmolarity of the order of 120 to 500 mOsm and preferably close to the isotonicity (290 ± 5mOsm), chosen from:
- sodium chloride NaCl;
- potassium chloride KCl;
- magnesium chloride MgCl 2 ;
- calcium chloride CaCl 2 ;
- anhydrous sodium sulphate Na 2 SO 4 ; this bottom salt can be included in the buffer system;
- at least one preservative, having antioxidant and / or antibiotic properties chosen from:
- 2-phenoxyethanol;
- parabens;
- BHT;
- isothiazolones (
Proclin® 150 or 300); - derivatives of imidazole or urea;
- antibiotics ;
- a natural antibiotic cellular penetration compound (ionophore) which further facilitates the penetration of the dye (s) chosen from:
- ionophore I to NH 4 + (nonatin);
- ionophore III with Ca 2+ (Calcimycin);
- the ionophore at Cl - ;
- Ionophore I to K + (Valinomycin).
Les composants selon l'invention sont récapitulés dans le tableau donné ci-après ainsi que des fourchettes de concentrations appropriées.
La présente divulgation propose également un appareil pour la mise en oeuvre du procédé selon l'invention qui est caractérisé par :
- un bac d'analyse adapté à recevoir ladite solution d'analyse ;
- un moyen de mesure par spectrophotométrie dans ledit bac du taux d'hémoglobine présent dans ladite solution d'analyse ;
- un moyen de prélèvement de ladite solution d'analyse ;
- un moyen de mesure optique sur ledit prélèvement afin de réaliser une analyse leucocytaire.
- an analysis bin adapted to receive said analysis solution;
- means for measuring spectrophotometrically in said tray the hemoglobin level present in said analysis solution;
- means for sampling said analysis solution;
- optical measuring means on said sample to perform a leukocyte analysis.
La présente divulgation vise un dispositif optique pour un automate d'analyse automatique d'un échantillon de sang, particulièrement avantageux en outre pour la mise en oeuvre du procédé selon le premier objet de l'invention.The present disclosure is directed to an optical device for an automaton for automatic analysis of a blood sample, which is particularly advantageous for the implementation of the method according to the first subject of the invention.
Comme mentionné plus haut, certaines sous-populations de leucocytes ne peuvent être différenciées que par des mesures optiques, par exemple une mesure de diffraction par la cellule à un ou plusieurs angles, ou, une mesure de l'absorbance de la cellule. Les systèmes optiques de caractérisation d'une cellule sanguine ont une base commune dans laquelle on retrouve une source lumineuse émettant un faisceau lumineux, une cuve optique dans laquelle les cellules sanguines traversent le faisceau lumineux, un système de réglage du faisceau lumineux sur le flux de cellules et des moyens de mesure de la lumière issue de la cuve optique après interception par les cellules. Notamment dans le cas de la caractérisation leucocytaire, les globules blancs se déplacent en un flux dans la cuve. Ils y sont éclairés par un faisceau lumineux focalisé sur le flux, que l'on appelle flux échantillon.As mentioned above, certain leukocyte subpopulations can be differentiated only by optical measurements, for example a diffraction measurement by the cell at one or more angles, or a measurement of the absorbance of the cell. Optical characterization systems of a blood cell have a common base in which we find a light source emitting a beam light, an optical tank in which the blood cells pass through the light beam, a light beam adjustment system on the flow of cells and means for measuring the light from the optical tank after interception by the cells. In particular in the case of leucocyte characterization, the white blood cells move in a flow in the tank. They are illuminated by a light beam focused on the flow, called sample flow.
De tels dispositifs sont coûteux ; en particulier, les lasers utilisés en tant que sources lumineuses, qui sont en outre encombrants et requièrent généralement un système de dissipation thermique ; les diodes laser, comme les lasers nécessitent des systèmes d'alignement coûteux. Les faisceaux lumineux émis par ces sources ont une répartition transverse de la lumière de forme sensiblement gaussienne. Ainsi, l'intensité n'est sensiblement constante et maximale que dans une partie étroite et centrale du rayon. Les systèmes d'alignement permettent d'aligner cette partie centrale avec le flux d'échantillon. En outre, la largeur du flux d'échantillon ne doit pas excéder celle de cette partie centrale, et la précision du système d'alignement doit être d'autant plus grande que ces deux largeurs sont proches. Il en résulte la nécessité de réduite au maximum la largeur du flux d'échantillon.Such devices are expensive; in particular, lasers used as light sources, which are also bulky and generally require a heat dissipation system; Laser diodes, like lasers, require expensive alignment systems. The light beams emitted by these sources have a transverse distribution of the light of substantially Gaussian form. Thus, the intensity is substantially constant and maximum only in a narrow and central part of the radius. Alignment systems align this central portion with the sample flow. In addition, the width of the sample flow must not exceed that of this central part, and the accuracy of the alignment system must be even greater than these two widths are close. This results in the need to minimize the width of the sample stream.
Le flux échantillon contenant les cellules sanguines à compter et/ou à différencier doit être d'autant plus étroit que la lumière est focalisée. Ainsi, on utilise un flux dont la largeur de la section est inférieure à 50µm, qui doit traverser le faisceau lumineux lui-même focalisé en un pinceau de section plus grande que celle du flux d'échantillon. Cela nécessite un système d'injection particulièrement précis, donc coûteux, du flux dans la cuve optique. Dans l'art antérieur, on obtient un tel résultat en utilisant un système de type hydrofocus (contraction de l'expression anglaise « hydrodynamic focusing »). On gaine le flux échantillon par un flux de manchonnage. Un injecteur pour le flux échantillon est immergé au coeur du flux de manchonnage. Le flux d'échantillon ainsi créé est étiré, ou focalisé, durant son trajet depuis l'injecteur jusqu'à la zone éclairée par le faisceau lumineux, de sorte qu'il a, à cet endroit, une largeur souhaitée d'environ 5 à 50µm de diamètre. Un simple ou un double manchonnage est parfois nécessaire pour atteindre cet objectif.The sample flow containing the blood cells to be counted and / or to be differentiated must be narrower the closer the light is focused. Thus, using a flux whose section width is less than 50 .mu.m, which must pass through the light beam itself focused in a brush section greater than that of the sample stream. This requires a particularly precise injection system, therefore expensive, the flow in the optical tank. In the prior art, such a result is obtained by using a hydrofocus-type system (contraction of the English expression "hydrodynamic focusing"). The sample flow is sheathed by a sleeve flow. An injector for the sample flow is immersed in the core of the sleeve flow. The sample stream thus created is stretched, or focused, during its journey from the injector to the area illuminated by the light beam, so that it has a desired width of about 5 to 50μm in diameter. A single or double sleeve is sometimes necessary to achieve this goal.
En outre, comme on l'a dit précédemment, compte tenu du niveau de précision requis, un système de réglage est indispensable pour faire coïncider le flux de cellules avec le faisceau lumineux. Deux approches sont possibles : on peut déplacer le flux de cellules ou le faisceau lumineux. Si l'on choisit de déplacer le flux de cellules sanguines, il faut déplacer l'ensemble de la cuve optique. Lorsque cette option est retenue, la cuve est montée sur une table à translation qui assure un déplacement fin et uniforme selon deux axes grâce à ses roulements à billes. Un tel ensemble mécanique de précision est assez onéreux. On peut également déplacer le faisceau lumineux pour le faire coïncider avec le flux de cellules sanguines. Ceci est généralement réalisé à l'aide de plusieurs prismes orientables. Cette solution, qui combine des éléments optiques à de la mécanique de précision, implique également un coût élevé.In addition, as mentioned above, given the level of precision required, an adjustment system is essential to make the flow of cells coincide with the light beam. Two approaches are possible: we can move the cell flow or the light beam. If you choose to move the flow of blood cells, you must move the entire optical tank. When this option is chosen, the tank is mounted on a translation table which ensures a smooth and uniform movement along two axes thanks to its ball bearings. Such a mechanical set of precision is quite expensive. It is also possible to move the light beam to coincide with the flow of blood cells. This is usually done using several orientable prisms. This solution, which combines optical elements with precision mechanics, also involves a high cost.
Par ailleurs, lorsqu'elle traverse le faisceau lumineux, la cellule sanguine dévie la trajectoire des rayons lumineux. L'intensité et l'angle des rayons déviés permettent d'obtenir des informations sur le type de la cellule. Deux gammes d'angles sont généralement utilisées : de petits angles inférieurs à dix degrés par rapport à l'axe optique et de grands angles sensiblement perpendiculaires à l'axe optique. Dans la gamme des petits angles, deux informations sont utiles : les pertes dans l'axe et la diffraction. Perpendiculairement à l'axe optique, on mesure généralement la diffusion et la fluorescence. Pour les deux gammes d'angles, la lumière doit donc être répartie dans deux canaux différents. Ceci est généralement réalisé par des miroirs dichroïques ou encore par des filtres interférentiels. Ces composants optiques sont tous deux réalisés par dépôts de couches minces sur un substrat de verre. Leur efficacité est bonne mais il existe une grande disparité d'un filtre à l'autre et leur durée de vie est limitée. Ils doivent donc être changés régulièrement.Moreover, when it passes through the light beam, the blood cell deviates the trajectory of the light rays. The intensity and the angle of the deviated rays make it possible to obtain information on the type of the cell. Two ranges of angles are generally used: small angles less than ten degrees from the optical axis and large angles substantially perpendicular to the optical axis. In the range of small angles, two pieces of information are useful: the losses in the axis and the diffraction. Perpendicular to the optical axis, diffusion and fluorescence are generally measured. For both ranges of angles, the light must be distributed in two different channels. This is usually done by dichroic mirrors or by interference filters. These optical components are both made by deposition of thin layers on a glass substrate. Their efficiency is good but there is a great disparity from one filter to another and their life is limited. They must therefore be changed regularly.
Tous ces dispositifs généralement encombrants, sont en outre fragiles et demandent un entretien, lui aussi très coûteux. De tels dispositifs sont donc réservés à des laboratoires d'analyses suffisamment importants pour pouvoir investir dans de tels automates.All these generally bulky devices are also fragile and require maintenance, which is also very expensive. Such devices are therefore reserved for analytical laboratories large enough to invest in such automata.
La divulgation a pour but de proposer un dispositif de différenciation leucocytaire et/ou le comptage leucocytaire plus simple et plus économique tant à fabriquer qu'à entretenir, permettant l'utilisation d'automates, ainsi équipés, par des laboratoires de moindre importance, tout en gardant une qualité de mesure suffisante.The purpose of the disclosure is to propose a leucocyte differentiation device and / or leucocyte counting that is simpler and more economical both to manufacture and to maintain, allowing the use of automated systems as well as equipped, by laboratories of less importance, while keeping a sufficient quality of measurement.
Dans la divulgation il est proposé un dispositif optique pour le comptage et/ou la différenciation leucocytaire dans un automate d'analyse sanguine, caractérisé en ce qu'il comprend une source lumineuse du type diode électroluminescente pour éclairer un échantillon sanguin qui circule dans la cuve optique selon un axe d'injection, à l'aide d'un faisceau lumineux source. Une telle diode permet d'obtenir un faisceau lumineux plus homogène sur la largeur de sa section, donc une zone de lecture plus large et homogène.In the disclosure there is provided an optical device for leukocyte counting and / or differentiation in a blood analysis machine, characterized in that it comprises a light-emitting diode-type light source for illuminating a blood sample which circulates in the tank. optical along an injection axis, using a source light beam. Such a diode makes it possible to obtain a more homogeneous light beam over the width of its section, thus a wider and homogeneous reading zone.
De préférence, la diode émet une lumière dont la longueur d'onde est inférieure à 600 nanomètres, et de façon encore plus préférée inférieure à 500 nanomètres. Une telle longueur d'onde permet un meilleur rendement de diffraction, donc une meilleure précision pour des mesures utilisant la diffraction.Preferably, the diode emits light whose wavelength is less than 600 nanometers, and even more preferably less than 500 nanometers. Such a wavelength allows a better diffraction efficiency, thus a better accuracy for measurements using diffraction.
En outre, la largeur du faisceau émis par le dispositif optique, c'est-à-dire le faisceau source qui éclaire le flux échantillon, est avantageusement comprise entre 50 et 200 microns (µm), au voisinage de l'axe d'injection, ce qui permet d'éclairer un flux échantillon plus large, tout en permettant une précision suffisante aux mesures effectuées. Encore plus avantageusement, cette largeur est comprise entre 90 et 120 microns. Une telle largeur de flux est notamment permise par l'utilisation de diodes électroluminescente.In addition, the width of the beam emitted by the optical device, that is to say the source beam which illuminates the sample flow, is advantageously between 50 and 200 microns (μm), in the vicinity of the injection axis. This allows a larger sample flow to be illuminated, while allowing sufficient precision to the measurements made. Even more advantageously, this width is between 90 and 120 microns. Such a flux width is notably allowed by the use of light emitting diodes.
De préférence, le faisceau lumineux source est émis sensiblement en direction de la cuve, sensiblement transversalement à la direction d'écoulement de l'échantillon. Une lame transparente prévue pour être traversée entre deux faces opposées par le faisceau source, montée en rotation et disposée entre la diode et la cuve peut permettre de déplacer transversalement le faisceau lumineux, grâce à sa double réfraction lors de la traversée de la lame. La rotation de la lame permet de modifier l'angle d'incidence du faisceau sur la lame, et ainsi de régler la valeur du décalage transversal. De préférence, la lame transparente est montée en rotation autour d'un axe sensiblement parallèle au déplacement de l'échantillon sanguin dans la cuve.Preferably, the source light beam is emitted substantially in the direction of the tank, substantially transversely to the flow direction of the sample. A transparent blade designed to be traversed between two opposite faces by the source beam, mounted in rotation and disposed between the diode and the tank can allow to move transversely the light beam, thanks to its double refraction during the crossing of the blade. The rotation of the blade makes it possible to modify the angle of incidence of the beam on the blade, and thus to adjust the value of the transverse offset. Preferably, the transparent blade is rotated about an axis substantially parallel to the displacement of the blood sample in the tank.
Au-delà de la cuve optique, on utilise avantageusement des moyens de séparation par pertes de Fresnel pour un faisceau lumineux résultant incident issu du faisceau lumineux source, séparant ainsi ledit faisceau en un faisceau résultant axial, et au moins un faisceau résultant de perte constitué par les pertes de Fresnel au passage des moyens de séparations. Les moyens de séparation comprennent au moins une surface de séparation qui est une surface d'un matériau de séparation transparent, le faisceau axial ayant traversé le matériau transparent et le faisceau issu des pertes de Fresnel ayant été réfléchi par la surface de séparation, ladite surface étant biaise relativement au faisceau lumineux au delà de la cuve. Une simple lame de verre, peu coûteuse, peut servir de moyens de séparation. En outre elle a une durée de vie quasiment illimitée et sans maintenance, contrairement aux miroirs dichroïques ou aux filtres interférentiels.Beyond the optical tank, Fresnel loss separation means are advantageously used for a resulting light beam. incident from the source light beam, thereby separating said beam into an axial resultant beam, and at least one resultant beam of loss constituted by Fresnel losses at the passage of the separation means. The separation means comprise at least one separation surface which is a surface of a transparent separation material, the axial beam having passed through the transparent material and the beam resulting from Fresnel losses having been reflected by the separation surface, said surface being biased relative to the light beam beyond the tank. A simple glass slide, inexpensive, can serve as a means of separation. In addition, it has an almost unlimited and maintenance-free life, unlike dichroic mirrors or interference filters.
Le dispositif peut comprendre en outre un appareil pour mesurer la lumière du faisceau résultant axial et au moins un autre appareil pour mesurer la lumière de l'au moins un faisceau issu des pertes de Fresnel. Ces appareils de mesure peuvent notamment comprendre des moyens pour une mesure parmi une mesure de la fluorescence, une mesure des pertes lumineuses au voisinage de l'axe et une mesure de la diffraction au voisinage de l'axe. Il peut en outre comprendre des moyens de mesure de la diffraction du faisceau lumineux aux grands angles par l'échantillon dans la cuve. A titre d'exemple, ces grands angles peuvent être des angles compris entre 60° et 150°.The device may further comprise an apparatus for measuring the light of the resulting axial beam and at least one other apparatus for measuring the light of the at least one beam resulting from Fresnel losses. These measurement devices can in particular comprise means for a measurement among a measurement of the fluorescence, a measurement of the light losses in the vicinity of the axis and a measurement of the diffraction in the vicinity of the axis. It may further comprise means for measuring the diffraction of the light beam at large angles by the sample in the tank. For example, these large angles may be angles between 60 ° and 150 °.
Le dispositif peut aussi comprendre, sur le trajet du faisceau avant la cuve, au moins un diaphragme d'arrêt pour une lumière parasite.The device may also comprise, in the path of the beam before the tank, at least one stop diaphragm for stray light.
La divulgation porte aussi sur un appareil d'hématologie, notamment un automate d'analyse sanguine équipé d'un tel dispositif.The disclosure also relates to a hematology apparatus, in particular a blood analysis machine equipped with such a device.
La présente divulgation vise également une cuve optique à circulation pour un dispositif optique adapté au comptage et à la différenciation leucocytaire, par exemple un cytomètre de flux, ainsi qu'un appareil d'analyse équipé d'une telle cuve. Le but de l'invention est de proposer une cuve plus simple et plus économique tant à fabriquer qu'à entretenir, permettant l'utilisation d'automates ainsi équipés par des laboratoires de moindre importance, tout en gardant une qualité de mesure suffisant.The present disclosure also relates to a circulating optical tank for an optical device suitable for leucocyte counting and differentiation, for example a flow cytometer, as well as an analysis apparatus equipped with such a tank. The object of the invention is to provide a simpler and more economical tank both to manufacture and maintain, allowing the use of automated machines and equipped by smaller laboratories, while maintaining a sufficient measurement quality.
Selon la divulgation, une cuve à circulation pour un dispositif optique pour le comptage et la différenciation leucocytaire dans un automate d'analyse sanguine, est caractérisée en ce que dans une zone d'analyse de la cuve (304), la section de la cuve a au moins une dimension transversale comprise entre 1 et 5 millimètres. Cette section peut être sensiblement rectangulaire et la dimension transversale être mesurée sur l'un et/ou l'autre des côtés du rectangle.According to the disclosure, a flow cell for an optical device for leukocyte counting and differentiation in an automated analyzer characterized in that in an analysis zone of the vessel (304), the section of the vessel has at least one transverse dimension of between 1 and 5 millimeters. This section may be substantially rectangular and the transverse dimension be measured on one and / or the other side of the rectangle.
Une telle cuve peut alors être réalisée, au moins partiellement, en un matériau plastique injecté. Une telle cuve est particulièrement avantageuse à fabriquer relativement aux cuves de l'art antérieur, généralement formée de parois en quartz assemblées par collage.Such a tank can then be made, at least partially, of an injected plastic material. Such a tank is particularly advantageous to manufacture relative to the tanks of the prior art, generally formed of quartz walls assembled by gluing.
La cuve peut en outre comprendre au moins une lentille moulée d'une seule pièce avec la cuve. Cette au moins une lentille peut comprendre une lentille prévue pour être disposée latéralement relativement à un axe optique. Elle peut comprendre une lentille hémisphérique.The tank may further comprise at least one lens molded integrally with the tank. This at least one lens may comprise a lens designed to be disposed laterally relative to an optical axis. It can include a hemispherical lens.
La cuve peut comprendre selon un axe optique, une fenêtre pour l'entrée d'un faisceau lumineux et une fenêtre pour la sortie du faisceau. Au moins une fenêtre peut être moulée d'une seule pièce avec cuve et/ou être un insert en une matière transparente, par exemple du quartz ou du verre.The tank may comprise, along an optical axis, a window for the entry of a light beam and a window for the exit of the beam. At least one window may be molded in one piece with a tank and / or be an insert made of a transparent material, for example quartz or glass.
La cuve peut avantageusement comprendre un injecteur pour un flux d'échantillon et des moyens pour former un flux de manchonnage autour du flux d'injection. L'injecteur peut comprendre un orifice de sortie dont le diamètre est compris entre 20 microns et 150 microns, permettant d'obtenir un flux d'échantillon sensiblement plus large que les flux de l'art antérieur. Contrairement aux dispositifs de l'art antérieur, ce n'est pas le flux de manchonnage qui impose la largeur du flux échantillon en l'étirant, mais la forme et la section de sortie de l'injecteur. Le flux de gainage n'a donc plus un rôle actif, mais seulement un rôle passif, notamment, par exemple, pour centrer le flux échantillon dans une cuve de grande largeur.The vessel may advantageously comprise an injector for a sample flow and means for forming a sleeving flow around the injection flow. The injector may comprise an outlet orifice whose diameter is between 20 microns and 150 microns, making it possible to obtain a sample flow that is substantially larger than the fluxes of the prior art. Unlike the devices of the prior art, it is not the sleeving flow that imposes the width of the sample flow by stretching it, but the shape and the outlet section of the injector. The sheath flow therefore no longer has an active role, but only a passive role, in particular, for example, to center the sample flow in a tank of great width.
Selon un premier mode de réalisation, cet injecteur peut être formé d'une seule pièce en un matériau sensiblement rigide. Ce matériau peut être, par exemple, un acier inoxydable, une céramique, du rubis synthétique ou une matière plastique ou plusieurs de ces matériaux.According to a first embodiment, this injector can be formed in one piece from a substantially rigid material. This material may be, for example, a stainless steel, a ceramic, synthetic ruby or a plastic or many of these materials.
Selon un deuxième mode de réalisation cet injecteur peut comprendre un tube de structure rigide, par exemple métallique, par exemple en acier inoxydable, et à l'intérieur du tube de structure, un tube de gainage en matériau plastique prolongé par un bec formé d'une seule pièce avec le tube de gainage. Le matériau plastique de l'injecteur peut être un polytétrafluoroéthylène, qui permet une circulation plus aisée de l'échantillon dans la tube et en réduit les risques l'encrassement.According to a second embodiment, this injector may comprise a tube of rigid structure, for example a metal structure, for example made of stainless steel, and inside the structural tube, a plastic cladding tube extended by a spout formed of one piece with the sheath tube. The plastic material of the injector may be a polytetrafluoroethylene, which allows easier movement of the sample in the tube and reduces the risk of fouling.
La divulgation porte en outre sur un injecteur pour une cuve selon la divulgation, injecteur réalisé selon l'un de ces modes de réalisation.The disclosure also relates to an injector for a tank according to the disclosure, an injector made according to one of these embodiments.
La divulgation porte aussi sur un appareil d'hématologie, notamment un automate d'analyse sanguine, équipé d'une cuve selon l'invention.The disclosure also relates to a hematology apparatus, in particular a blood analysis machine, equipped with a tank according to the invention.
La présente divulgation vise également un dispositif hydraulique pour un appareil d'analyse hématologique, qui soit simplifié et plus économique tant à fabriquer qu'à entretenir et qui permette l'utilisation d'automates équipés d'un tel dispositif par des laboratoires de moindre importance, tout en gardant une qualité de mesure suffisante. La présente divulgation vise aussi un procédé d'analyse adapté à un tel dispositif.The present disclosure also relates to a hydraulic device for a hematological analysis device, which is simplified and more economical both to manufacture and to maintain and which allows the use of automated devices equipped with such a device by laboratories of lesser importance. , while keeping a sufficient quality of measurement. The present disclosure also relates to an analysis method adapted to such a device.
La présente divulgation propose ainsi un dispositif hydraulique pour un appareil d'analyse sanguine, notamment un automate, comprenant des moyens pour injecter sous pression un flux d'échantillon dans une cuve optique à circulation et pour créer un flux de manchonnage liquide autour du flux d'échantillon, avec un liquide de manchonnage, caractérisé en ce qu'il comprend des moyens pour réguler un débit du flux d'échantillon relativement au débit du liquide de manchonnage. Une telle régulation peut permettre de maintenir des flux homogènes et sensiblement non turbulents dans la cuve.The present disclosure thus proposes a hydraulic device for a blood analysis apparatus, in particular an automaton, comprising means for injecting under pressure a stream of sample into a circulating optical tank and to create a liquid sleeve flow around the flow of the sample. sample, with a sleeving liquid, characterized in that it comprises means for regulating a flow rate of the sample flow relative to the flow rate of the sleeving liquid. Such a regulation can make it possible to maintain homogeneous and substantially non-turbulent flows in the tank.
Les moyens d'injection peuvent comprendre des seringues, un circuit hydraulique et des électrovannes. Ces moyens peuvent comprendre des moyens pour injecter l'échantillon sous pression relativement au flux de manchonnage.The injection means may comprise syringes, a hydraulic circuit and solenoid valves. These means may comprise means for injecting the sample under pressure relative to the sleeve flow.
Ce dispositif peut avantageusement comprendre des moyens pour former un piston pour l'échantillon injecté avec un liquide de pistonnage. Un tel liquide de pistonnage permet de n'utiliser qu'un petit échantillon suffisant pour l'analyse, le reste du liquide nécessaire pour l'injection étant un liquide disponible dans l'appareil d'analyse, et moins précieux que l'échantillon.This device may advantageously comprise means for forming a piston for the sample injected with a liquid piston. Such a sweeping liquid makes it possible to use only a small sample sufficient for the analysis, the remainder of the liquid necessary for the injection being a liquid available in the analysis apparatus, and less valuable than the sample.
Le manchonnage est particulièrement avantageux pour utiliser une cuve de grande section tout en maintenant une section réduite pour le flux d'échantillon. Parmi les moyens de régulation du flux d'échantillon relativement au flux de manchonnage, le dispositif peut avantageusement comprendre des moyens pour réguler un débit du liquide de pistonnage relativement au débit du liquide de manchonnage. Les moyens de régulation peuvent comprendre des moyens de perte de charge dans un circuit de dérivation pour le liquide de pistonnage et/ou des moyens de perte de charge dans un circuit de dérivation pour le liquide de manchonnage. Par exemple, les moyens de perte de charge peuvent êtres choisis parmi une longueur connue d'un tuyau calibré, une résistance hydraulique fixe et une résistance variable.Sleeving is particularly advantageous for using a large section tank while maintaining a reduced section for the flow sample. Among the means for regulating the sample flow relative to the sleeve flow, the device may advantageously comprise means for regulating a flow rate of the sweeping liquid relative to the flow rate of the sleeving liquid. The regulating means may comprise means of pressure drop in a bypass circuit for the piston liquid and / or pressure loss means in a bypass circuit for the sleeving liquid. For example, the pressure drop means may be chosen from a known length of a calibrated pipe, a fixed hydraulic resistance and a variable resistance.
Le dispositif hydraulique peut ne comprendre qu'une seule motorisation, par exemple un seul moteur électrique, pour générer simultanément le flux d'échantillon et le flux de manchonnage. En outre, il peut comprendre au moins deux seringues pour générer le flux d'échantillon et le flux de manchonnage, les pistons des seringues étant rigidement liés entre eux. Ils sont ainsi animés d'un mouvement commun et les flux d'échantillon et de manchonnage sont bien simultanés.The hydraulic device may comprise only one motor, for example a single electric motor, to simultaneously generate the sample flow and the sleeve flow. In addition, it may comprise at least two syringes to generate the sample flow and the sleeve flow, the syringe pistons being rigidly interconnected. They are thus animated by a common movement and the sample and sleeving flows are simultaneous.
Notamment, une cuve à hydrofocus de l'art antérieur peut être utilisée avec un circuit tel que précédemment décrit, l'injection de l'échantillon dans cette cuve pouvant se faire sans pression relativement au fluide de manchonnage.In particular, a hydrofocus tank of the prior art can be used with a circuit as previously described, the injection of the sample into this tank can be done without pressure relative to the sleeving fluid.
Selon la divulgation, il est aussi proposé un procédé d'analyse d'un échantillon sanguin dans un cytomètre à circulation, caractérisé en ce que l'on injecte, éventuellement sous pression, un échantillon sanguin dans une cuve à circulation du cytomètre, l'échantillon y formant un flux d'échantillon et on crée un flux de manchonnage liquide autour du flux d'échantillon, avec un liquide de manchonnage, caractérisé en ce qu'on régule le débit du flux d'échantillon relativement au débit du liquide de manchonnage.According to the disclosure, there is also provided a method of analyzing a blood sample in a flow cytometer, characterized in that a blood sample is injected, possibly under pressure, into a flow cell of the cytometer, the sample forming a sample flow and creating a liquid sleeve flow around the sample stream, with a sleeving liquid, characterized in that the flow rate of the sample flow is regulated relative to the flow of the sleeving liquid .
En particulier, on peut introduire l'échantillon dans une branche d'injection d'un circuit hydraulique, et introduire en amont de l'échantillon dans la branche d'injection, un liquide de pistonnage, le liquide de pistonnage servant à pousser l'échantillon lors de son injection dans la cuve. Ce liquide de pistonnage peut être choisi parmi un réactif et un diluant, de préférence un réactif. Il n'est donc pas utile de prévoir un autre liquide que ceux strictement nécessaires pour la préparation de l'échantillon en vue de son ou de ses analyses.In particular, it is possible to introduce the sample into an injection branch of a hydraulic circuit, and to introduce upstream of the sample into the injection branch, a piston liquid, the piston liquid used to push the sample when it is injected into the tank. This slurry liquid may be selected from a reagent and a diluent, preferably a reagent. It is therefore not necessary to provide another than those strictly necessary for the preparation of the sample for its analysis.
On peut aussi créer autour du flux d'échantillon dans la cuve, un flux de manchonnage avec un liquide de manchonnage. Ce liquide de manchonnage peut aussi être choisi parmi un réactif et un diluant, de préférence un diluant. Là aussi, Il n'est donc pas utile de prévoir un autre liquide que ceux strictement nécessaires pour la préparation de l'échantillon en vue de son ou de ses analyse.It is also possible to create around the sample flow in the tank, a sleeve flow with a sleeving liquid. This sleeving liquid can also be selected from a reagent and a diluent, preferably a diluent. Again, it is not necessary to provide a liquid other than those strictly necessary for the preparation of the sample for his or her analysis.
Dans le cas de mise en oeuvre d'un procédé d'hydrofocus, ou d'une cuve, il est avantageux de réguler le débit du liquide de pistonnage relativement au débit du liquide de manchonnage, par exemple en introduisant une perte de charge dans un circuit de dérivation pour un liquide de pistonnage et/ou des moyens de perte de charge dans un circuit de dérivation pour un liquide de manchonnage.In the case of implementation of a hydrofocus process, or a tank, it is advantageous to regulate the flow of the liquid sweeping relative to the flow of the sleeving liquid, for example by introducing a pressure drop in a branch circuit for a plunger and / or pressure drop means in a branch circuit for a sleeve fluid.
Dans un procédé utilisant une cuve, on peut aisément prévoir que l'échantillon du sang présente un taux de dilution d'au moins 1/100ème. En effet, dans un tel procédé, on peut introduire l'échantillon sous pression relativement au fluide de manchonnage, dans la cuve, à une vitesse supérieure à celle des procédés de l'art antérieur, et avec des largeurs de sections supérieures pour le flux d'échantillon dans la cuve. Ainsi, sans augmenter le temps d'analyse, on peut pour une différenciation et un comptage des globules blancs utiliser un taux de dilution identique à celui pratiqué habituellement pour la mesure de l'hémoglobine, en particulier des taux de dilution sensiblement compris entre 1/100ème et 1/500ème, particulièrement entre 1/160ème et 1/180ème.In a method using a vat, it can easily be expected that the blood sample has a dilution ratio of at least 1 / 100th. Indeed, in such a method, it is possible to introduce the sample under pressure relative to the sleeving fluid, into the tank, at a speed greater than that of the processes of the prior art, and with widths of higher sections for the flow. sample in the tank. Thus, without increasing the analysis time, it is possible for differentiation and counting of white blood cells to use a dilution ratio identical to that customarily used for the measurement of hemoglobin, in particular dilution levels substantially between 1 / 100 th and 1/500 th, particularly between 1/160 th and 1/180 th.
La divulgation concerne aussi un appareil d'hématologie, notamment un automate d'analyse sanguine, caractérisé en ce qu'il comprend un dispositif hydraulique selon l'invention.The disclosure also relates to a hematology apparatus, particularly a blood analysis machine, characterized in that it comprises a hydraulic device according to the invention.
La présente invention sera mieux comprise et d'autres avantages apparaîtront à la lumière de la description qui va suivre d'exemples de réalisation, description faite notamment en référence aux dessins annexés sur lesquels :
- la
figure 1 illustre de manière schématique un exemple d'appareillage ; - les
figures 2a-2e sont des graphes de test de linéarité de la mesure de l'hémoglobine par spectrophotométrie selon le procédé de l'invention; - les
figures 2f-2i sont des cytographes correspondants ; - la
figure 3 est une vue schématique d'un automate d'analyse d'un échantillon de sang mettant en oeuvre un dispositif hydraulique ; - la
figure 4 est une vue schématique longitudinale d'ensemble d'un dispositif optique ; - la
figure 5 est une vue schématique longitudinale plus détaillée du dispositif optique de lafigure 4 , dans un plan perpendiculaire à celui de lafigure 4 ; - la
figure 6 est une vue en perspective d'une cuve optique ; - la
figure 7 est une vue en coupe longitudinale d'un premier mode de réalisation d'un injecteur pour une cuve optique ; - la
figure 8 est une vue en coupe longitudinale d'un deuxième mode de réalisation d'un injecteur pour une cuve optique ; - la
figure 9 est une vue en coupe longitudinale d'une extrémité de l'injecteur de lafigure 8 ; - la
figure 10 est une vue en coupe longitudinale d'une cuve illustrant un procédé d'injection de l'art antérieur pour l'échantillon de sang dans la cuve ; et - la
figures 11a-11c sont des graphes illustrant les résultats obtenus avec un automate mettant en oeuvre le procédé de l'invention et utilisant un cytographe avec le dispositif et la cuve optiques.
- the
figure 1 schematically illustrates an example of apparatus; - the
Figures 2a-2e are linearity test graphs of the measurement of hemoglobin by spectrophotometry according to the method of the invention; - the
Figures 2f-2i are corresponding cytographs; - the
figure 3 is a schematic view of an automated analysis of a blood sample using a hydraulic device; - the
figure 4 is a schematic longitudinal overall view of an optical device; - the
figure 5 is a more detailed longitudinal schematic view of the optical device of thefigure 4 , in a plane perpendicular to that of thefigure 4 ; - the
figure 6 is a perspective view of an optical tank; - the
figure 7 is a longitudinal sectional view of a first embodiment of an injector for an optical tank; - the
figure 8 is a longitudinal sectional view of a second embodiment of an injector for an optical tank; - the
figure 9 is a longitudinal sectional view of one end of the injector of thefigure 8 ; - the
figure 10 is a longitudinal sectional view of a vessel illustrating a prior art injection method for the blood sample in the vessel; and - the
Figures 11a-11c are graphs illustrating the results obtained with an automaton implementing the method of the invention and using a cytograph with the device and the optical tank.
Sur la
L'appareillage fonctionne de la manière suivante :
- une aliquote de sang (15,6 µl) est injectée dans la cuve d'analyse 1 et diluée avec 2 ml de diluant de manière à former une pré-solution d'analyse ; le taux de dilution est de 1/130e ;
- une fraction très faible (
environ 20 µl) est prélevée sur cette pré-solution d'analyse et déposée dans lebac 9 de comptage des globules rouges et des plaquettes ; - 0,7 ml de réactif est alors ajouté au reliquat dans le bac d'analyse 1 : la lyse dure environ 10 secondes (pour détruire les globules rouges, former et stabiliser le complexe oxyhémoglobine), la solution d'analyse ainsi formée présente un taux de dilution
final d'environ 1/173e ; une fraction de ladite solution d'analyse est prélevée et injectée dans la cuve optique 7 où peut avoir lieu l'analyse des globules blancs (comptage et/ou différenciation leucocytaires par sous populations) ; simultanément dans le bac d'analyse 1, les globules blancs sont comptés par une mesure de résistivité et l'hémoglobine par une mesure par absorbance à la longueur d'onde du complexe oxyhémoglobine formé.
- an aliquot of blood (15.6 μl) is injected into the
analysis vat 1 and diluted with 2 ml of diluent to form a pre-solution of analysis; the dilution ratio is 1/130 e ; - a very small fraction (approximately 20 .mu.l) is taken from this pre-solution of analysis and deposited in the
tank 9 for counting red blood cells and platelets; - 0.7 ml of reagent is then added to the residue in the analysis tank 1: the lysis lasts about 10 seconds (to destroy the red blood cells, form and stabilize the oxyhemoglobin complex), the analysis solution thus formed has a rate of final dilution of about 1/173 e ; a fraction of said analysis solution is removed and injected into the
optical tank 7 where the analysis of the white blood cells (leucocyte counting and / or differentiation by subpopulations) can take place; simultaneously in thetest tray 1, the white blood cells are counted by a resistivity measurement and the hemoglobin by an absorbance measurement at the wavelength of the formed oxyhemoglobin complex.
Un dispositif optique, particulièrement adapté à une analyse leucocytaire sur une solution d'analyse ayant un taux de dilution inférieur à 1/100e est décrit plus loin, plus particulièrement adapté à une dilution comprise entre 1/160e et 1/180e. Par convention, on considère qu'un taux de dilution de 1/160e est inférieur à un taux de 1/100e.An optical device, particularly suitable for leucocyte analysis on an analysis solution having a dilution ratio of less than 1/100 e is described later, more particularly adapted to a dilution between 1/160 e and 1/180 e . By convention, it is considered that a dilution ratio of 1/160 e is lower than a rate of 1/100 e .
Il va de soi que des variantes de réalisation du procédé et de l'appareillage décris ci-dessus sont possibles :
- pour l'appareillage : on peut prévoir des moyens pour introduire séparément le composé de lyse, le composé leucoprotecteur et le composé stabilisateur du complexe formé avec l'hémoglobine dans la cuve d'analyse 1, et donc plus sous la forme d'un mono-réactif ; les moyens 6 pour mesurer la résistivité de la solution d'analyse sont optionnels, le nombre global de globules blancs pouvant être obtenus par l'analyse optique de la solution d'analyse ; de même pour le bac de comptage 9 et les moyens de mesure 10 de la résistivité dans ce bac peuvent être prévus uniquement si l'on souhaite une analyse complète de l'échantillon de sang ;
- de même pour le procédé : on peut prévoir l'introduction des composés réactionnels de manière indépendante ou groupée à la place d'un mono-réactif, l'introduction pouvant être menée de manière simultanée ou successivement ; on peut omettre l'étape préalable de comptage des globules rouges et des plaquettes et l'étape de comptage global des globules blancs ; par ailleurs, on peut réaliser deux dilutions successives de l'échantillon de sang : une première dilution particulièrement adaptée à une différenciation leucocytaire (
environ au 1/80e) telle qu'elle est menée dans les cytomètres classiques connus de type hydrofocus, sur laquelle est prélevée la fraction nécessaire à cette différenciation leucocytaire, puis dans un second temps une deuxième dilution adaptée à une mesure de l'hémoglobine (compriseentre 1/100eet 1/500e) telle qu'elle est possible avec les spectromètres connus.
- for the apparatus: it is possible to provide means for separately introducing the lysing compound, the leucoprotective compound and the stabilizing compound of the complex formed with hemoglobin into the
analysis vessel 1, and therefore no longer in the form of a mono -reactive; themeans 6 for measuring the resistivity of the analysis solution are optional, the overall number of white blood cells being obtainable by the optical analysis of the analysis solution; similarly for thecounting tank 9 and themeans 10 for measuring the resistivity in this tank can be provided only if it is desired a complete analysis of the blood sample; - the same applies to the process: the introduction of the reaction compounds can be provided independently or in groups in place of a mono-reagent, the introduction being able to be carried out simultaneously or successively; it is possible to omit the prior step of counting red blood cells and platelets and the step of global counting of white blood cells; moreover, two successive dilutions of the blood sample can be carried out: a first dilution which is particularly suitable for leucocyte differentiation (approximately 1/80 e ) as carried out in the known conventional hydrofocus cytometers, on which the fraction necessary for this leucocyte differentiation is taken, then in a second time a second dilution adapted to a measurement of hemoglobin (between 1/100 e and 1/500 e ) as it is possible with the known spectrometers.
Selon une autre variante encore, le bac 1 peut servir dans un second temps à faire le comptage des globules rouges et des plaquettes après nettoyage, en remplissant le bac avec un prélèvement en attente dans une aiguille d'une seringue.According to another variant, the
On va maintenant décrire les résultats obtenus avec un exemple particulier de (mono)réactif :
- Un monoréactif est préparé à partir de la formulation Eosinofix® de la société ABX commercialisée pour la détermination leucocytaire en cytométrie de flux et contenant à cet effet un composé pour lyser les globules rouges et un composé leucoprotecteur (cf le brevet
EP0430750
- A monoreactant is prepared from the Eosinofix® formulation of the company ABX marketed for the leucocyte determination in flow cytometry and containing for this purpose a compound for lysing red blood cells and a leukoprotective compound (see patent
EP0430750
Des tests de linéarité ont été menés à l'aide d'un spectrophotomètre à 542nm. Les graphes sont donnés aux
- la
figure 2a correspond à une lyse de référence pour la mesure de l'hémoglobine par spectrophotométrie (LMG® vendue par la société ORPHEE); - les
figures 2b, 2c et2d correspondent au monoréactif selon l'exemple de réalisation N°4 avec respectivement comme agent de stabilisation du complexe de l'hémoglobine le Tiron, le DDAPS et l'imidazole ; et - la
figure 2e correspond au procédé de l'invention mis en oeuvre en utilisant comme monoréactif de l'éosinofix® seul, c'est-à-dire ne contenant pas d'agent de stabilisation du complexe de l'hémoglobine conformément à la présente invention.
- the
figure 2a corresponds to a reference lysis for the measurement of hemoglobin by spectrophotometry (LMG® sold by ORPHEE); - the
Figures 2b, 2c and2d correspond to the mono-reagent according to the example of embodiment No. 4 with respectively stabilizing agent of the hemoglobin complex Tiron, DDAPS and imidazole; and - the
figure 2e corresponds to the process of the invention implemented using eosinofix® alone as monoreactive, that is to say not containing stabilizing agent of the hemoglobin complex according to the present invention.
Pour les trois essais effectués selon l'invention, on obtient bien un test de linéarité positif avec pour chacun un coefficient de corrélation R2 de 1 ± 10-4 (reporté sur la figure). Ce résultat est conforme à celui obtenu avec la lyse de référence de la
En revanche, comme on le voit sur la
Les
On peut aussi se référer au cytographe de la
On va maintenant décrire le dispositif hydraulique.We will now describe the hydraulic device.
La
L'automate illustré à la
Un autre dispositif d'analyse 120 est plus particulièrement dédié au comptage et à la différenciation des globules blancs, par exemple sur le tout ou la partie de l'échantillon prélevé dans le bac 102. Par la suite nous appèlerons aussi échantillon ce tout ou cette partie. Le dispositif d'analyse des globules blancs 120 comprend notamment un dispositif optique 200 et une cuve optique 300. La cuve optique est reliée au bac 102 via le circuit hydraulique 100.Another
Un jeu de seringue permet le déplacement des liquides dans le circuit hydraulique. Parmi ces seringues, une seringue 105 dédiée au diluant et une seringue 106 dédiée au réactif sont représentées pour la bonne compréhension du dispositif. D'autres seringues qui ne sont pas représentées car elles ne sont pas nécessaires à la compréhension du dispositif, peuvent compléter le dispositif.A syringe set allows the movement of liquids in the hydraulic circuit. Among these syringes, a
Outre des conduites pour la circulation des liquides, le circuit hydraulique comprend des électrovannes pour la commutation de différents circuits dans le circuit hydraulique 100, selon l'utilisation qui en est faite à un moment donné de l'analyse. Huit électrovannes 111-119 parmi les électrovannes du circuit hydraulique 100 sont illustrées à la
La conception du circuit hydraulique tel qu'il sera décrit par la suite, permet de n'utiliser qu'une seule motorisation M pour les seringues illustrées. La même motorisation peut d'ailleurs être utilisée pour d'autres seringues. Ainsi, les pistons des seringues 105,106 sont rigidement reliés entre eux. Ils ont donc un mouvement simultané, soit en poussée P, lorsqu'ils s'enfoncent dans le cylindre respectif de chaque seringue, ou en traction T lorsqu'ils en sont extraits.The design of the hydraulic circuit as will be described later allows to use only one motorization M for syringes illustrated. The same motorization can be used for other syringes. Thus, the pistons of the
On va maintenant décrire la disposition puis le fonctionnement hydraulique de l'automate.We will now describe the arrangement and the hydraulic operation of the automaton.
La cuve 300 comprend un corps extérieur 301 et un injecteur 302, à l'intérieur du corps 301, un volume de manchonnage 303 est formé entre le corps et l'injecteur.The
Le circuit hydraulique 100 comprend :
- une branche d'injection 131 qui s'étend en amont de l'injecteur, entre l'injecteur et la vanne 111 ;
- une branche d'échantillon 132 qui se raccorde en
un branchement d'échantillon 142 sur la branche d'injection et s'étend jusqu'aubac 102 ; - une branche d'aspiration 133 qui se raccorde en
un branchement d'aspiration 143 sur la branche d'injection, en amont du branchement d'échantillon 142, viala vanne 113 et s'étend jusqu'à une source de vide 107, par exemple une seringue ou une pompe péristaltique ; - une branche de décharge 134 qui se raccorde en un branchement de décharge 144 sur la branche d'injection, en amont du branchement d'aspiration 143, et s'étend jusqu'à la réserve de produit de réactif 104 ;
- une branche de manchonnage 135 qui s'étend en amont du corps 301 et relie le volume de manchonnage et la vanne 115 ;
- une branche de
dilution 136 qui s'étend entre la vanne 116et une utilisation 108 pour le diluant via la vanne 115; - une branche de diluant 137 qui s'étend entre la réserve de diluant 103 et la vanne 116 ;
- une branche de réactif 140 qui s'étend entre la réserve de réactif 104 et la vanne 117 ;
- une branche de réaction 141 qui s'étend entre la vanne 117
et une utilisation 109 pour le réactif via la vanne 111; - une branche de vidange 138 pour
le bac 102 qui s'étend entre lebac 102 et la source de vide 107 viala vanne 118, la branche d'échantillon 132 étant reliée avec la branche de vidange entre lebac 102 etla vanne 118, et la branche d'aspiration étant reliée avec la branche de vidange 132 au-delà de la vanne 118 relativement au bac; et, - une branche de sortie 139 qui relie l'aval de la cuve 300 , via
la vanne 119, avec un bac à déchet, par exemple à la pression atmosphérique où via une source d'aspiration, une seringue ou une pompe péristaltique.
- an
injection branch 131 which extends upstream of the injector, between the injector and the valve 111; - a
sample branch 132 which connects to asample branch 142 on the injection branch and extends to thetray 102; - a
suction branch 133 which connects to asuction branch 143 on the injection branch, upstream of thesample branch 142, via thevalve 113 and extends to avacuum source 107, through example a syringe or a peristaltic pump; - a
discharge branch 134 which connects to adischarge branch 144 on the injection branch, upstream of thesuction branch 143, and extends to thereagent stock 104; - a
sleeve branch 135 which extends upstream of thebody 301 and connects the sleeve volume and thevalve 115; - a
dilution branch 136 extending between thevalve 116 and ause 108 for the diluent via thevalve 115; - a
diluent branch 137 which extends between thediluent supply 103 and thevalve 116; - a
reagent branch 140 extending between thereagent reservoir 104 and thevalve 117; - a
reaction branch 141 which extends between thevalve 117 and ause 109 for the reagent via the valve 111; - a
drain branch 138 for thetank 102 which extends between thetank 102 and thevacuum source 107 via thevalve 118, thesample branch 132 being connected with the drain branch between thetank 102 and thevalve 118, and the suction branch being connected with thedrain branch 132 beyond thevalve 118 relative to the tank; and, - an
output branch 139 which connects the downstream of thetank 300, via thevalve 119, with a waste container, for example at atmospheric pressure or via a suction source, a syringe or a peristaltic pump.
Dans une première position 116A de la vanne 116, la seringue de dilution 105 est en communication avec la réserve de diluant, de sorte qu'une traction T permet de remplir la seringue 105 avec du diluant.In a
La seringue de dilution contenant du diluant, dans un premier cas, la vanne 116 étant dans sa deuxième position 116B qui relie la seringue 105 avec la branche de dilution 136 et la vanne 115 étant dans une première position 115A qui relie la branche de dilution avec l'utilisation 108 pour le diluant, une poussée P permet de déplacer le diluant jusqu'à cette utilisation 108, par exemple dans le bac 102, par exemple pour une dilution de l'échantillon global.The dilution syringe containing diluent, in a first case, the
Dans un deuxième cas, la vanne 116 étant dans sa deuxième position 116B et la vanne 115 étant dans sa deuxième position 115B qui relie la branche de dilution avec la branche de manchonnage 135, une poussée P permet de déplacer le diluant jusque dans la cuve optique 300, pour y former un flux de manchonnage. L'utilité de ce flux de manchonnage dans le cadre de l'invention sera analysée lors d'une description ultérieure de la cuve 300.In a second case, the
La vanne 117 étant dans une première position 117A qui relie la seringue de réactif avec la réserve 104 de réactif, et la vanne 114 étant dans une première position 114A qui coupe la branche de décharge 134, une traction T permet de remplir la seringue de réactif 106 avec du réactif.Since the
La seringue de réactif contenant du réactif, dans un premier cas, la vanne 117 étant dans sa deuxième position 117B qui relie la seringue de réactif 104 avec la branche réaction 141 et la vanne 111 étant dans une première position 111A qui relie la branche de réaction avec l'utilisation 109 pour le réactif, une poussée P permet de déplacer le réactif jusqu'à cette utilisation 109, par exemple dans le bac 102, par exemple pour une lyse de l'échantillon global.The reagent syringe containing the reagent, in a first case, the
Dans un deuxième cas, la vanne 117 étant dans sa deuxième position 117B et la vanne 111 étant dans sa deuxième position 111B qui relie la branche de réaction 141 avec la branche de d'injection 131, la seringue de réactif 106 est directement reliée à l'injecteur 302.In a second case, the
La vanne 118 étant dans une première position 118A qui isole la branche d'aspiration 133 d'avec la branche d'échantillon 132 au travers de la branche de vidange, la vanne 112 étant dans une première position 112A qui relie l'amont à l'aval de la branche d'échantillon 132, la vanne 113 étant dans une première position 113A qui relie l'aval à l'amont de la branche d'aspiration 133, donc à la source de vide 107, l'échantillon à analyser est aspiré jusque dans la branche d'injection 131, entre le branchement d'échantillon 142 et le branchement d'aspiration 143.The
La branche de décharge 134 comprend une résistance fluidique 150 variable ou calibrée.The
Lorsque la seringue de diluant 105 contient du diluant, que la seringue de réactif 106 contient du réactif et qu'un échantillon sanguin à analyser se trouve dans la branche d'injection 131 ; lorsqu'en outre les vannes 112,113 sont dans leurs deuxièmes positions 112B,113B qui isolent l'amont et l'aval de leurs branches respectives ; lorsqu'encore les vannes 115,116 sont dans leurs deuxièmes positions 115B,116B qui relient la seringue 105 de diluant avec le volume de manchonnage 303; lorsqu'enfin les vannes 111,117 sont dans leurs deuxièmes positions 111B,117B qui relient la seringue de réactif 106 avec l'injecteur 302 et que la vanne 114 est dans sa deuxième position 114B ; un seul mouvement de poussée P généré par la seule motorisation M, permet de propulser le diluant, le réactif et l'échantillon de sang en direction et au travers de la cuve 300, alors qu'une partie du réactif, fonction de la résistance fluidique 150, est retournée dans la réserve de réactif 104.When the
La résistance 150 permet notamment de régler les débits relatifs des liquides de manchonnage et de pistonnage. Cela permet d'adapter ces débits aux différentes fonctions de ces liquides. En particulier, cela permet d'avoir des vitesses d'écoulement similaires pour le manchonnage et l'échantillon dans la zone d'analyse 304 lorsque l'on utilise une cuve à hydrofocus classique.The
Notamment, la branche de décharge 134 et les dispositions précédemment décrites permettent l'utilisation d'une unique motorisation, donc de réduire notablement le coût d'un automate d'analyse, ainsi que son encombrement.In particular, the
Le diluant forme dans une zone d'analyse 304 de la cuve 300 un flux de manchonnage pour l'échantillon (voir particulièrement les
Bien sûr, d'autres seringues, vannes et branches, non représentées à la
On va maintenant décrire le dispositif optique 200, particulièrement en regard des
Le dispositif optique comprend une source 201 de lumière sensiblement monochrome. Cette source de lumière est une diode électroluminescente. La lumière est émise principalement selon un axe optique X200. L'axe optique X200 est disposé sensiblement perpendiculairement à un axe d'injection X300 de déplacement de l'échantillon dans la cuve optique 300. Les deux axes X200 et X300 définissent ensemble un plan optique.The optical device comprises a
Pour éviter que le faisceau lumineux source 311 issus de la source 201 ne soit pas pollué par des lumières parasites, un jeu de trois diaphragmes est disposé chacun perpendiculairement sur le parcours du faisceau. Les diaphragmes 202 sont percés d'orifices dont le diamètre est sensiblement égal au faisceau et progressivement augmenté dans chaque diaphragme pour s'adapter au diamètre du faisceau à mesure où ce diamètre augmente en s'éloignant de la source 201. Le faisceau traverse ensuite un dispositif de focalisation 203 constitué par une ou plusieurs lentilles.To prevent the source
Au-delà du dispositif de focalisation le faisceau rencontre un dispositif de réglage qui permet de déplacer l'axe optique dans un plan perpendiculaire à l'axe d'injection X300, c'est-à-dire transversalement relativement au déplacement de l'échantillon dans la cuve. Un décalage latéral du faisceau peut conduire à un éclairage partiel ou nul de l'échantillon ce qui a une influence directe sur le résultat de l'analyse.Beyond the focusing device, the beam encounters an adjustment device which makes it possible to move the optical axis in a plane perpendicular to the injection axis X300, that is to say transversely relative to the displacement of the sample. in the tank. A lateral shift of the beam can lead to partial or no illumination of the sample which has a direct influence on the result of the analysis.
Dans le cadre de l'exemple décrit, le dispositif de réglage est constitué d'une lame transparente 220 montée en rotation autour d'un axe X220. L'axe 221 est sensiblement parallèle à l'axe d'injection X300. Si la lame est disposée perpendiculairement à l'axe optique X200, le faisceau la traverse sans être dévié. Par contre, si la lame forme un angle avec l'axe optique, une double réfaction, à l'entrée et à la sortie de la lame, décale le faisceau dans un plan perpendiculaire à l'axe de réglage X220. L'axe de réglage X220 étant sensiblement parallèle à l'axe d'injection X300, seul un décalage transversal est généré par la réfraction dans la lame. Le décalage est d'autant plus grand que l'épaisseur et/ou l'indice de réfraction de la lame sont grands et que la lame est inclinée relativement à l'axe optique. Ainsi, pour une lame d'épaisseur et d'indice de réfraction choisis, il suffit de faire tourner la lame 220 autour de son axe X220 pour régler la position du faisceau relativement à l'échantillon qui se déplace dans la zone d'analyse 304 de la cuve optique 300. Un tel dispositif de réglage est particulièrement économique relativement aux dispositifs de l'art antérieur, d'autant qu'une rotation précise est généralement plus facile à mettre en oeuvre qu'une translation précise, utilisant de la mécanique de grande précision.In the context of the example described, the adjustment device consists of a
Après avoir pénétré la cuve et traversé l'échantillon, le faisceau source 211 devient au moins partiellement un faisceau résultant axial 212, qui sort de la cuve sensiblement selon l'axe optique. Le faisceau résultant axial 212 est porteur d'informations sur l'échantillon qu'il vient de traverser.After penetrating the vessel and passing through the sample, the
Pour permettre des mesures simultanées de plusieurs de ces informations on doit pouvoir analyser le faisceau avec plusieurs appareils de mesure 222,223. En particulier, l'analyse optique repose sur la détection de la lumière diffractée selon deux gammes d'angles : petits et grands angles. Dans chacune des gammes d'angles, on utilise deux informations différentes. Il convient donc de répartir la lumière dans deux canaux différents pour chaque gamme. On utilise donc des moyens 205 pour séparer le faisceau résultant 212 en deux faisceaux résultants 213,214. Les moyens de séparation sont principalement constitués d'une lame séparatrice 205. Cette lame séparatrice est une lame transparente en verre. Elle est disposée à 45 degrés de l'axe optique. Il en résulte un faisceau résultant axial secondaire 213, formé par la lumière ayant traversé la lame séparatrice, et un faisceau résultant de perte 214 formé par les pertes de Fresnel, c'est-à-dire par la lumière réfléchie par la lame séparatrice.To allow simultaneous measurements of several of these information it is necessary to be able to analyze the beam with several measuring
Une telle lame séparatrice est d'un coût très faible relativement aux moyens de séparation utilisés dans l'art antérieur dans les dispositifs d'analyse optique de ce type. En particulier, du fait qu'elle ne comprend aucun revêtement réfléchissant ajouté, elle est quasiment inaltérable dans le temps et ne nécessite sensiblement aucun entretien. Compte tenu des réflexions multiples à l'intérieur de la lame et tenant compte de la polarisation du rayonnement incident du faisceau résultant axial, entre 5 et 15% de l'énergie est réfléchie, le reste étant transmis sous la forme du faisceau résultant axial secondaire.Such a separating blade is of a very low cost relative to the separation means used in the prior art in optical analysis devices of this type. In particular, because it does not include any reflective coating added, it is almost unalterable over time and requires substantially no maintenance. Given the multiple reflections inside the plate and taking into account the polarization of the incident radiation of the resulting axial beam, between 5 and 15% of the energy is reflected, the remainder being transmitted in the form of the resulting secondary axial beam .
Entre la cuve et la lame séparatrice, le faisceau résultant axial 212 est rendu parallèle par des moyens adaptés 206. Au-delà de la lame séparatrice, les faisceaux résultants 213,214 sont de nouveau focalisés par des moyens adaptés respectifs 207,208, en vue de leur analyse par les appareils de mesure respectifs 222,223.Between the vessel and the separator blade, the resulting
Dans l'exemple décrit, l'appareil de mesure 222, qui analyse le faisceau résultant axial secondaire 213 est un appareil de mesure de la diffraction au voisinage de l'axe optique par les cellules sanguines (dite mesure du FSC). Dans l'exemple décrit, l'appareil de mesure 223, qui analyse le faisceau issu des pertes de Fresnel 214 est un appareil de mesure des pertes lumineuses dans l'axe (dite mesure de l'ALL), c'est-à-dire de l'occultation de la lumière par les cellules dans l'échantillon.In the example described, the measuring
La
Notamment en référence à la
La cuve 350 de la
La cuve 300, illustrée aux
Le corps est réalisé en un matériau injecté, de préférence un matériau plastique. Un tel procédé de fabrication permet d'obtenir des formes complexes. En particulier, une lentille 305 est moulée dans le corps. Cette lentille permet de collecter la lumière occultée, diffractée ou diffusée par les cellules sanguines.The body is made of an injected material, preferably a plastic material. Such a manufacturing method makes it possible to obtain complex shapes. In particular, a
Cette lentille doit avoir des dimensions, notamment un diamètre suffisant pour que les éventuelles inhomogénéités locales dans le matériau injecté soient négligeables relativement à ces dimensions. Dans l'exemple illustré, la lentille 305 à un diamètre voisin de 3mm. Cette lentille injectée est une lentille latérale 305 traversé par le faisceau résultant latéral 315. En outre, la lentille latérale doit permettre de collecter la lumière selon le maximum de directions, c'est-à-dire avec un champ directionnel maximal, Ainsi, plus la lentille est près de l'échantillon, plus le champ directionnel est large. Dans l'exemple illustré, la lentille est une lentille hémisphérique, dite à 90°. De plus, la lentille étant une partie de la paroi de la cuve, il y a directement contact avec le liquide dans la cuve, c'est-à-dire qu'il n'y a pas de lame d'air, à faible indice de réfraction, entre l'échantillon et la lentille. La mesure en est d'autant améliorée.This lens must have dimensions, including a sufficient diameter so that any local inhomogeneities in the injected material are negligible relative to these dimensions. In the illustrated example, the
Pour s'affranchir des défauts d'homogénéité, là où la lumière est particulièrement focalisée on utilise du verre, par exemple un verre de type BK7. C'est notamment le cas pour les fenêtres axiales 306, là où le faisceau source 211 pénètre dans la cuve et là où le faisceau résultant axial 212 en sort.To overcome homogeneity defects, where the light is particularly focused glass is used, for example a glass type BK7. This is particularly the case for the
Pour pouvoir réaliser une lentille injectée d'une telle dimension il convient donc que dans la zone d'analyse, la cuve 300 ait des dimensions au moins comparables. En outre ces grandes dimensions permettent d'intégrer des fenêtres en verre dans des parois en plastique, alors que dans les cuves de l'art antérieur, de petites dimensions, celles-ci sont faites de parois entièrement de verre ou de quartz. Dans l'exemple illustré notamment aux
En amont de la zone d'analyse 304, le corps 301 de la cuve enveloppe l'injecteur 302 et forme autour de l'injecteur le volume de manchonnage 303. Les parois de l'injecteur séparent un flux 311 formé par l'échantillon, à l'intérieur de l'injecteur d'avec un flux de manchonnage 312, dans le volume de manchonnage. Le flux d'échantillon est issu de la branche d'injection 131 du circuit hydraulique 100. Le flux de manchonnage est issu de la branche de manchonnage 135 du circuit hydraulique. Dans la zone d'analyse, les deux flux sont en contact, restent concentriques et s'écoulent simultanément dans la cuve.Upstream of the
Afin de réduire les coûts de fabrication de l'automate, il peut être avantageux de réduire la précision de fabrication des pièces. Comme cela a été dit plus haut, un tel but peut être atteint en créant un flux d'échantillon de plus grande section.In order to reduce the manufacturing costs of the machine, it may be advantageous to reduce the manufacturing precision of the parts. As mentioned above, such a goal can be achieved by creating a larger sample stream.
Cependant, si l'on utilise une technique de l'art antérieur où le flux d'échantillon est étiré par un flux de manchonnage, un flux d'échantillon de grande section sera turbulent, ce qui nuirait notablement à la précision des mesures. En outre le flux d'échantillon serait progressivement réduit en section, ce qui va à l'encontre de l'effet recherché, qui est d'avoir un flux d'échantillon de grande section. Un tel but est atteint grâce à la mise en oeuvre du circuit hydraulique 100, précédemment décrit en référence à la
En outre, un injecteur 302 tel qu'illustré à la
Un injecteur 302 tel qu'illustré à la
Le bec a une section progressivement rétrécie depuis un diamètre intérieur D321 du tube de gainage jusqu'à un diamètre intérieur D323 d'un orifice de sortie 323 à une extrémité aval 324 du bec 322. Dans l'exemple décrit, l'extrémité aval 324 est un cylindre de longueur L324. La paroi du bec est d'abord concave vers l'intérieur, puis infléchie pour devenir intérieurement convexe, la section du bec étant ainsi progressivement rétrécie depuis l'amont vers l'aval, depuis le diamètre D321 jusqu'au diamètre D324. La surface concave est tangente avec la surface intérieure du tube de gainage cylindrique. La surface convexe est tangente avec la surface intérieure de l'extrémité cylindrique 324. Dans l'exemple décrit, le diamètre D323 de l'orifice 323 est d'environ 60 microns, le diamètre intérieur D321 du tube de gainage est d'environ 1 millimètre, la longueur L322 du bec est d'environ 2,5 millimètres, celle L320 du tube de structure d'environ 6 millimètres et celle de l'extrémité cylindrique L324 d'environ 200 microns.The spout has a gradually narrowed section from an inside diameter D321 of the cladding tube to an inside diameter D323 of an
Un injecteur 302 tel qu'illustré aux
Le bec se rétrécit progressivement intérieurement, depuis un diamètre intérieur D331 pour le tube 331, jusqu'à un diamètre intérieur D333 d'un orifice de sortie 333 pour l'échantillon, à une extrémité aval 334 du bec 332. Dans, l'exemple illustré, le rétrécissement se fait selon un tronc de cône ouvert d'un angle compris, de préférence, entre 9 et 10 degrés. Au-delà du tronc de cône est jusqu'à l'orifice de sortie 333, le diamètre reste constant dans une partie cylindrique 335, de longueur L335 et de diamètre D333.The beak gradually narrows internally, from an inside diameter D331 for the
A l'extérieur du bec, le diamètre extérieur en est progressivement moins réduit selon un tronc de côte ouvert d'un angle compris entre 8 et 9 degrés environ, puis, dans l'extrémité nettement plus réduit selon un tronc de côte ouvert d'un angle A334 compris entre 35 et 45 degrés environ, jusqu'à un diamètre extérieur D334 autour de l'orifice de sortie 333. D334 est environ 3 à 4 plus grand que D333.Outside the beak, the outer diameter is progressively less reduced along an open coast trunk at an angle of between 8 and 9 degrees, then, in the much smaller end along an open coast trunk. an angle A334 of between about 35 and 45 degrees, to an outer diameter D334 around the
A titre d'exemple D333=60µm, D334=200µm et A334=40°.By way of example, D333 = 60 μm, D334 = 200 μm and A334 = 40%.
Grâce aux différents dispositifs précédemment décrits, on peut obtenir une vitesse d'injection élevée. Cette dans l'exemple décrit, il est possible d'injecté un échantillon de plus de 200 microlitres en moins de 10 secondes. En particulier, une telle vitesse d'injection permet d'utiliser un taux de dilution important de l'échantillon de sang, sans augmenter la durée de l'analyse relativement à des automates de l'art antérieur. En particulier, une même dilution, par exemple 1/160ème, peut être utilisé pour l'analyse de l'hémoglobine par le dispositif 110 (voir
Les
Ces figures montrent que grâce à l'invention, on peut réaliser une analyse au moins du taux d'hémoglobine, du taux de globules blancs et une différentiation leucocytaire en utilisant la formulation défini dans les revendisations, notamment sous forme de mono-réactif.These figures show that, thanks to the invention, it is possible to perform at least a hemoglobin level, white blood cell count and leucocyte differentiation analysis using the formulation defined in the claims, in particular in the form of a mono-reagent.
Bien sûr, l'invention n'est pas limitée aux exemples qui viennent d'être décrits et de nombreux aménagements peuvent être apportés à ces exemples sans sortir du cadre de l'invention.Of course, the invention is not limited to the examples that have just been described and many adjustments can be made to these examples without departing from the scope of the invention.
Par exemple, d'autres produits que le diluant ou le réactif peuvent être utilisés pour respectivement former le flux de manchonnage et le piston fluide, particulièrement s'ils sont disponibles dans l'automate notamment pour d'autres utilisations.For example, other products than the diluent or the reagent can be used to respectively form the sleeving flow and the fluid piston, particularly if they are available in the automat especially for other uses.
Aussi, au lieu d'être uniquement disposée sur le circuit d'injection, une résistance fluidique peut être disposée sur le circuit de manchonnage ou sur les deux à la fois. Ceci peut être fait en fonction du débit maximum donné par les moyens de déplacement des liquides destinés respectivement au pistonnage ou au manchonnage.Also, instead of being only disposed on the injection circuit, a fluidic resistor can be disposed on the sleeve circuit or both. This can be done as a function of the maximum flow rate given by the liquid displacement means intended respectively for swaging or sleeving.
Plusieurs ou toutes les lentilles de la cuve optique et/ou du dispositif optique peuvent aussi être réalisées injectées avec le corps de la cuve, au lieu d'une seule tel que précédemment illustré. En particulier, les fenêtres en verre peuvent être injectées. Particulièrement si les inhomogénéités dans le matériau injecté sont sensiblement négligeables en regard de la précision voulue pour les mesures.Several or all the lenses of the optical tank and / or the optical device may also be made injected with the body of the tank, instead of one as previously illustrated. In particular, the glass windows can be injected. Especially if the inhomogeneities in the injected material are substantially negligible compared to the accuracy required for measurements.
Un dispositif de réglage et/ou les moyens séparateurs précédemment décrits peuvent être utilisés indépendamment les uns des autres et éventuellement avec une autre source lumineuse qu'une diode électroluminescente.An adjusting device and / or the separating means described above may be used independently of one another and possibly with another light source than a light-emitting diode.
Claims (15)
- Method for the automatic analysis of a blood sample in which:* an analysis solution is formed in a single dilution and analysis tank, the analysis solution containing:- said blood sample,- a diluent,- a mono-reagent comprising at least one compound to lyse the erythrocytes, at least one compound to stabilize the haemoglobin in the form of oxyhaemoglobin and at least one compound to protect the leucocytes, allowing the distinguishing of at least five main leucocyte sub-populations, said compound to protect the leucocytes being chosen from betaines and sulphobetaines of quaternary ammoniums, tertiary amine oxides, glycosidic type compounds and.glucidic type compounds,* the haemoglobin level is measured in this analysis solution by spectrophotometry in said tank after the lysis of the erythrocytes; and* an appropriate quantity of this analysis solution is taken from said tank on which a leucocytic differentiation of at least five sub-populations is carried out by an optical means.
- Method according to claim 1, characterized in that a dye or mixture of dyes specifically labelling at least one leucocyte sub-population is also added to the mono-reagent.
- Method according to one of claims 1 to 2, characterized in that the mono-reagent is suitable to carry out the function of diluent in order to produce a mono-dilution of the blood sample.
- Method according to one of claims 1 to 3, characterized in that the analysis solution has a dilution rate of the blood sample suitable for measuring the haemoglobin by spectrophotometry.
- Method according to claim 4, characterized in that the dilution rate of the analysis solution is comprised between 1/100th and 1/500th, and preferably between 1/160th and 1/180th.
- Method according to claim 1, characterized in that the sampling of an appropriate quantity of analysis solution to carry out the leucocyte differentiation is carried out before the measurement of the haemoglobin in said tank, the dilution rate of the sampled measuring solution being suitable for said measurement by said optical means, and in that diluent is added to said remaining analysis solution so as to increase the dilution in said tank in order to obtain a dilution suitable for a measurement of the haemoglobin by spectrophotometry.
- Method according to claim 1, characterized in that the optical means is a hydrofocus-type flow cytometer, and the dilution rate of the sampled analysis solution for measurement by cytometry is less than 1/100th.
- Method according to claim 1, characterized in that the optical means is a cytometer with passive sleeving, and the dilution rate of the sampled solution for measurement by cytometry is greater than 1/100th.
- Method according to claim 7 or 8, characterized in that the dilution rate of the measuring solution for the measurement of the haemoglobin by spectrophotometry is comprised between 1/100th and 1/500th.
- Method according to any one of the previous claims, characterized in that in a stage before the addition of said compounds, a fraction of a pre-solution is taken constituted by the sample of blood and diluent on which counting of the erythrocytes and/or platelets is carried out by resistivity.
- Method according to any one of the previous claims, characterized in that lysis compound is an ionic surfactant chosen from:- the quaternary ammonium salts, preferably alkyltrimethylammonium salts and still more particularly cetyl-, dodecyl-, tetradecyl- and hexadecyltrimethylammonium bromides and chlorides;- pyridinium salts;- long-chain ethoxylated amines; and- alkyl sulphates (SDS).
- Method according to any one of the previous claims, characterized in that the compound which stabilizes the haemoglobin in the form of a oxyhaemoglobin complex is preferably chosen from:- mono or polydentate chelates presenting ligand atoms (non-binding pairs: O, N, S, and carboxy COO- groups etc.),- the aromatic compounds (mono or polydentate chelates) comprising ligand atoms (having non-binding pairs: O, N, S etc.), and- saponins, tertiary amine oxides, betaines and sulphobetaines of quaternary ammoniums.
- Method according to any one of the previous claims, characterized in that a fixing agent of the membrane of the leucocytes is also added to the analysis solution.
- Method according to any one of the previous claims, characterized in that a pH buffer capable of setting the pH of the analysis solution between 5.0 and 8.0, preferably at a pH of 7, is also added to the analysis solution.
- Method according to any one of the previous claims, characterized in that a background salt is also added to the analysis solution to ensure its conductivity.
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FR0503118A FR2883972B1 (en) | 2005-03-31 | 2005-03-31 | METHOD FOR ANALYSIS OF A BLOOD SAMPLE AND APPARATUS AND REAGENT FOR ITS IMPLEMENTATION |
PCT/FR2006/000622 WO2006103334A2 (en) | 2005-03-31 | 2006-03-22 | Method for analyzing a blood sample and apparatus and reagent for implementing said method |
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Families Citing this family (21)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
FR2883971B1 (en) | 2005-03-31 | 2007-11-16 | C2 Diagnostics Sa | OPTICAL BLOOD ANALYSIS DEVICE, ANALYZING APPARATUS EQUIPPED WITH SUCH A DEVICE |
CN102533933A (en) * | 2010-12-15 | 2012-07-04 | 潘申权 | Method, reagent and kit for detecting oral inflammatory index, and applications thereof |
WO2011097962A1 (en) * | 2010-02-12 | 2011-08-18 | Pan Shenquan | Method for detecting and tool for clearing stomatitis spots and stomatitis nodes |
JP5868982B2 (en) | 2010-09-16 | 2016-02-24 | ザ ジェネラル ホスピタル コーポレイション | Red blood cell dynamics for diagnosis |
KR101893219B1 (en) * | 2011-08-10 | 2018-08-29 | 엘지전자 주식회사 | Body fluid analyzing method and body fluid analyzing system using the same |
EP3865875A1 (en) * | 2011-09-25 | 2021-08-18 | Labrador Diagnostics LLC | Systems and methods for multi-analysis |
BR112015021902B1 (en) * | 2013-03-15 | 2021-06-15 | Iris International, Inc | LIQUID FOR INTRACELLULAR PARTICLE AND ORGANELA ALIGNMENT |
CN103472034B (en) * | 2013-08-23 | 2015-10-28 | 深圳中科强华科技有限公司 | A kind of blood cell analysis chip, analyser and analytical approach |
FR3034198B1 (en) * | 2015-03-26 | 2019-05-31 | C2 Diagnostics | HYDROFOCUS APPARATUS WITH SINGLE ANALYSIS SOLUTION |
KR102111903B1 (en) * | 2015-07-06 | 2020-05-15 | 후지필름 가부시키가이샤 | Blood analysis method and blood testing kit |
WO2017106461A1 (en) | 2015-12-15 | 2017-06-22 | The General Hospital Corporation | Methods of estimating blood glucose and related systems |
CN105716917B (en) * | 2016-01-22 | 2020-01-31 | 杭州奥泰生物技术股份有限公司 | collecting liquid for protecting and stabilizing hemoglobin in feces and application thereof |
WO2017177192A1 (en) | 2016-04-07 | 2017-10-12 | The General Hospital Corporation | White blood cell population dynamics |
CN106370472B (en) * | 2016-10-13 | 2023-06-27 | 浙江天行健水务有限公司 | Quantitative device for water quality available chlorine on-line detection equipment |
RU2663732C1 (en) * | 2017-05-04 | 2018-08-09 | Игорь Михайлович Рулев | Method of automatic selection and packaging of microbiological objects |
US11519921B2 (en) | 2018-05-17 | 2022-12-06 | Beckman Coulter, Inc. | Green concentrated reagent for hematology systems |
CN109735484B (en) * | 2018-12-12 | 2022-07-12 | 中元汇吉生物技术股份有限公司 | Cell protective agent, reagent and application of reagent |
FR3091924B1 (en) | 2019-01-23 | 2021-10-15 | Erba Diagnostics Ltd | Method for optimizing the concentration of elements of interest for visual measurements on a biological sample |
DE102019118171A1 (en) * | 2019-07-04 | 2021-01-07 | Endress+Hauser Conducta Gmbh+Co. Kg | Method of operating an automatic analyzer and an automatic analyzer |
CN112444619B (en) * | 2019-08-30 | 2023-12-19 | 深圳迈瑞动物医疗科技股份有限公司 | Impedance method counting device and blood cell analyzer |
CN114279778A (en) * | 2021-12-04 | 2022-04-05 | 南京岚煜生物科技有限公司 | Preparation method of composite high-value reference product |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5510267A (en) * | 1989-05-15 | 1996-04-23 | Abbott Laboratories | Flow cytometry lytic agent and method enabling 5-part leukocyte differential count |
Family Cites Families (13)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4529705A (en) * | 1983-06-06 | 1985-07-16 | Coulter Electronics, Inc. | Reagent for combined diluting and lysing whole blood |
SU1469464A1 (en) | 1987-03-20 | 1989-03-30 | Институт фотобиологии АН БССР | Method of determining erythrocyte resistivity |
JP2836865B2 (en) * | 1989-10-23 | 1998-12-14 | 東亜医用電子株式会社 | Reagents for measuring leukocytes and hemoglobin in blood |
FR2654744B1 (en) * | 1989-11-20 | 1992-03-13 | Abx Sa | REAGENT AND METHOD OF USING SAME FOR THE AUTOMATIC DETERMINATION IN FLOW CYTOMETRY OF AT LEAST ONE LEUKOCYTAIC SUB-POPULATION FROM TOTAL BLOOD. |
JPH04172234A (en) * | 1990-11-05 | 1992-06-19 | Hitachi Ltd | Cell analyzer |
JP3529786B2 (en) * | 1994-07-14 | 2004-05-24 | アボット・ラボラトリーズ | Cyanide-free assay method and reagents for hemoglobin and leukocytes in whole blood |
US5786224A (en) * | 1995-06-29 | 1998-07-28 | Coulter Corporation | Reagent and method for differential determination of leukocytes in blood |
JP3545852B2 (en) * | 1995-09-20 | 2004-07-21 | 株式会社堀場製作所 | Automatic blood cell counter and liquid level detection method thereof |
JP3830613B2 (en) * | 1997-04-18 | 2006-10-04 | シスメックス株式会社 | Reagent for measuring leukocyte and hemoglobin concentration in blood |
FR2782166B1 (en) * | 1998-08-04 | 2000-10-06 | Abx Sa | REAGENT FOR MEASURING HEMOGLOBIN AND DETERMINING LEUKOCYTES IN A BLOOD SAMPLE |
BR0009420A (en) * | 1999-03-31 | 2002-04-09 | Bayer Ag | Single-channel, single-dilution detection method |
FR2821428B1 (en) * | 2001-02-23 | 2004-08-06 | Abx Sa | REAGENT AND METHOD FOR THE IDENTIFICATION AND COUNTING OF BIOLOGICAL CELLS |
JP4474099B2 (en) * | 2002-12-20 | 2010-06-02 | アークレイ株式会社 | Liquid storage container and cartridge |
-
2005
- 2005-03-31 FR FR0503118A patent/FR2883972B1/en active Active
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Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5510267A (en) * | 1989-05-15 | 1996-04-23 | Abbott Laboratories | Flow cytometry lytic agent and method enabling 5-part leukocyte differential count |
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MX2007011991A (en) | 2007-12-13 |
FR2883972B1 (en) | 2007-11-16 |
MA29386B1 (en) | 2008-04-01 |
BRPI0609493A2 (en) | 2010-04-13 |
KR101256250B1 (en) | 2013-04-22 |
EP1866651A2 (en) | 2007-12-19 |
ATE467837T1 (en) | 2010-05-15 |
US20090142744A1 (en) | 2009-06-04 |
WO2006103334A3 (en) | 2007-03-01 |
CN101185001B (en) | 2016-03-16 |
KR20070116680A (en) | 2007-12-10 |
AR052731A1 (en) | 2007-03-28 |
RU2007140259A (en) | 2009-05-10 |
US7981681B2 (en) | 2011-07-19 |
CN101185001A (en) | 2008-05-21 |
FR2883972A1 (en) | 2006-10-06 |
TW200724919A (en) | 2007-07-01 |
DE602006014260D1 (en) | 2010-06-24 |
RU2417378C2 (en) | 2011-04-27 |
JP2008534946A (en) | 2008-08-28 |
ZA200708616B (en) | 2008-10-29 |
WO2006103334A2 (en) | 2006-10-05 |
JP4838300B2 (en) | 2011-12-14 |
PL1866651T3 (en) | 2010-10-29 |
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